term paper on protein synthesis

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14.3 The Mechanism of Protein Synthesis

Protein synthesis.

As with mRNA synthesis, protein synthesis can be divided into three phases:  initiation, elongation, and termination . The process of translation is similar in prokaryotes and eukaryotes. Here we’ll explore how translation occurs in  E. coli , a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

Initiation of Translation

Protein synthesis begins with the formation of an  initiation complex . In  E. coli , this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special  initiator tRNA , called tRNA Metf .

The small subunit of the ribosome is first to bind to the mRNA template at a specific sequence called the Shine-Dalgarno sequence. The initiator tRNA then interacts with the start codon AUG. This tRNA carries the amino acid methionine, which is formylated after its attachment to the tRNA. The formylation creates a “faux” peptide bond between the formyl carboxyl group and the amino group of the methionine. Binding of the fMet-tRNA Metf  is mediated by the initiation factor IF-2. The fMet begins every polypeptide chain synthesized by  E. coli , but it is usually removed after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNA Met . After the formation of the initiation complex, the 30S ribosomal subunit is joined by the 50S subunit to form the translation complex. In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, eukaryotic IFs, and nucleoside triphosphates (GTP and ATP). The methionine on the charged initiator tRNA, called Met-tRNA i , is not formylated. However, Met-tRNA i  is distinct from other Met-tRNAs in that it can bind IFs.

Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5′ end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5′ cap. Once at the cap, the initiation complex tracks along the mRNA in the 5′ to 3′ direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to  Kozak’s rules , the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5′-gccRccAUGG-3′. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNA i , mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.

Translation, Elongation, and Termination

In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of  E. coli . When the translation complex is formed, the tRNA binding region of the ribosome consists of three compartments. The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. The initiating methionyl-tRNA, however, occupies the P site at the beginning of the elongation phase of translation in both prokaryotes and eukaryotes.

During translation elongation, the mRNA template provides tRNA binding specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the anticodon of the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically and randomly (?).

Elongation proceeds with charged tRNAs sequentially entering and leaving the ribosome as each new amino acid is added to the polypeptide chain. Movement of a tRNA from A to P to E site is induced by conformational changes that advance the ribosome by three bases in the 3′ direction. The energy for each step along the ribosome is donated by elongation factors that hydrolyze GTP. GTP energy is required both for the binding of a new aminoacyl-tRNA to the A site and for its translocation to the P site after formation of the peptide bond. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by  peptidyl transferase , an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from the high-energy bond linking each amino acid to its tRNA. After peptide bond formation, the ribosome advances relative to the mRNA and tRNAs such that the A-site tRNA that now holds the growing peptide chain will be present in the P site, and the P-site tRNA that is now uncharged moves to the E site and is expelled from the ribosome. Amazingly, the E. coli  translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino-acid protein can be translated in just 10 seconds.

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by  protein release factors  that resemble tRNAs. The releasing factors in both prokaryotes and eukaryotes instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Protein Folding, Modification, and Targeting

During and after translation, individual amino acids may be chemically modified, signal sequences appended, and the new protein “folded” into a distinct three-dimensional structure as a result of intramolecular interactions. A  signal sequence  is a short sequence at the amino end of a protein that directs it to a specific cellular compartment. These sequences can be thought of as the protein’s “train ticket” to its ultimate destination, and are recognized by signal-recognition proteins that act as conductors. For instance, a specific signal sequence terminus will direct a protein to the mitochondria or chloroplasts (in plants). Once the protein reaches its cellular destination, the signal sequence is usually clipped off.

Many proteins fold spontaneously, but some proteins require helper molecules, called  chaperones , to prevent them from aggregating during the complicated process of folding. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly.

College Biology I Copyright © by Melissa Hardy is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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16 Protein Synthesis Overview

Andrea Bierema

Learning Objectives

Students will be able to:

• Describe the structure and purpose of DNA and RNA.
• Describe the general process of protein synthesis.
• Describe the molecular anatomy of genes and genomes.
• Identify DNA and mRNA bases and binding patterns.
• Interpret a codon-amino acid chart.
• Given a DNA sequence, determine the corresponding mRNA sequence and amino acid sequence.

Central Dogma

The central dogma of molecular biology is that DNA codes for RNA and RNA codes for protein. In addition to DNA coding for RNA, much of the DNA regulates the synthesis of RNA- which ultimately means that it regulates the synthesis of protein. We will learn about protein synthesis regulation in a later chapter.

Protein synthesis consists of two main processes: transcription and translation. During the process of transcription —which occurs in the nucleus—an mRNA molecule is created by reading the DNA. Note that DNA never “becomes” RNA; rather, the DNA is “read” to make an RNA molecule. The mRNA leaves the nucleus and then, through the process of translation , the mRNA is read to create an amino acid sequence which folds into a protein.

Consider what the terms “transcribe” and “translate” mean in relation to language. To “transcribe” something means to rewrite text again in the same language while to “translate” something means to rewrite the text in a different language. Similar to these meanings, in biology, DNA is transcribed into RNA: both DNA and RNA are made of nucleic acid (i.e., the same “language”). With the assistance of proteins, DNA is “read” and transcribed into an mRNA sequence. To read RNA and create protein, though, we refer to it as being translated: RNA is made of nucleic acid and protein is made of amino acid (i.e., different “languages”). Therefore, DNA is transcribed to create an mRNA sequence, and then the mRNA sequence is translated to make a protein.

DNA and RNA

The two main types of nucleic acids are  deoxyribonucleic acid (DNA)  and  ribonucleic acid (RNA) . DNA is the genetic material in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope.

The cell’s entire genetic content is its genome, and the study of genomes is genomics. In eukaryotic cells but not in prokaryotes, a DNA molecule may contain tens of thousands of genes. Many genes contain information to make protein products (e.g., mRNA). Other genes code for RNA products. DNA controls all of the cellular activities by turning the genes “on” or “off.”

The other type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus but instead use an intermediary to communicate with the rest of the cell. This intermediary is the  messenger RNA (mRNA) . Other types of RNA—like rRNA, tRNA, and microRNA—are involved in protein synthesis and its regulation.

DNA and RNA are comprised of monomers that scientists call  nucleotides . The nucleotides combine with each other to form a  polynucleotide , DNA or RNA. Three components comprise each nucleotide: a nitrogenous base, a pentose (five-carbon) sugar, and a phosphate group. Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to one or more phosphate groups. Therefore, although the terms “base” and “nucleotide” are sometimes used interchangeably, a nucleotide contains a base as well as part of the sugar-phosphate backbone.

Comparison of RNA (left molecule) and DNA (right molecule). The color of the bases in RNA and DNA aligns with the colored boxes next to each base molecule.

Examine the image above and then answer the following questions:

What is a Gene?

The gene is the basic physical unit of inheritance. Genes are passed from parents to offspring and contain the information needed to specify traits. Genes are arranged, one after another, on structures called chromosomes. A chromosome contains a single, long DNA molecule, only a portion of which corresponds to a single gene. Humans have approximately 20,000 genes arranged on their chromosomes. Watch the following video for an animated view on the relationship between chromosomes and genes.

Protein Synthesis Overview

The two main processes in protein synthesis are transcription and translation. The following is an overview of each of these processes. Each process will be described in more detail in future chapters.

Transcription

A gene is complex: it contains not only the code for the resulting protein but also several regulatory factors that determine if and when the region that codes for a protein are read to create protein. What follows is a diagram of the components of a gene that are used in transcription.

For this chapter, we focus on the DNA and the ending product of transcription: mRNA.

Given a specific DNA strand, what is the sequence of the resulting mRNA molecule? We will learn about how mRNA is created in a later chapter.

Translation

Translation involves different types of RNA, and we will explain them in more detail in a later chapter: rRNA, tRNA, mRNA, and microRNA.

After an mRNA is created, it leaves the nucleus and is attracted to or attracts a ribosome, which is a molecule made of rRNA and polypeptides. Then, in the ribosome, and with the assistance of tRNAs, the mRNA is read and an amino acid sequence is created.

DNA and mRNA create sequences with just four types of bases; yet, these bases code for 20 unique amino acids (the makeup of protein). How is this possible? Watch the following video to find out!

The mRNA is read in sets of three bases known as codons. Each codon codes for a single amino acid. In this way, the mRNA is read and the protein product is made.

Below is a chart showing which codons code for which bases. There are two representations; move to the next slide for the second representation.

These charts can be a little confusing at first. Watch the following video to learn how to interpret both chart formats.

This chapter focused on DNA, mRNA, and protein sequences. The next several chapters describe the processes that take place during protein synthesis. Master how sequences are read during protein synthesis (the focus of the current chapter) before moving on to the next chapter. Below are some sources to help further your understanding!

Check out Learn.Genetics’ “How a Firefly’s Tail Makes Light” video for an overview of protein synthesis!

Need a little more practice?

Try out Learn.Genetics’ “Transcribe and Translate a Gene” and The Concord Consortium’s “ DNA to Protein ” interactives for further practice!

Attributions

This chapter is a modified derivative of the following articles:

“ Gene ” by National Human Genome Research Institute, National Institutes of Health, Talking Glossary of Genetic Terms. 

“Nucleic Acids” by OpenStax College,  Biology 2e , CC BY 4.0. Download the original article at https://openstax.org/books/biology-2e/pages/3-5-nucleic-acids

Media Attributions

• Central Dogma © Andrea Bierema
• DNA and RNA © Sponk is licensed under a CC BY-SA (Attribution ShareAlike) license

An Interactive Introduction to Organismal and Molecular Biology Copyright © 2021 by Andrea Bierema is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

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The Cell: A Molecular Approach. 2nd edition.

Chapter 7 protein synthesis, processing, and regulation.

Transcription and RNA processing are followed by translation , the synthesis of proteins as directed by mRNA templates. Proteins are the active players in most cell processes, implementing the myriad tasks that are directed by the information encoded in genomic DNA . Protein synthesis is thus the final stage of gene expression. However, the translation of mRNA is only the first step in the formation of a functional protein. The polypeptide chain must then fold into the appropriate three-dimensional conformation and, frequently, undergo various processing steps before being converted to its active form. These processing steps, particularly in eukaryotes, are intimately related to the sorting and transport of different proteins to their appropriate destinations within the cell.

Although the expression of most genes is regulated primarily at the level of transcription (see Chapter 6), gene expression can also be controlled at the level of translation , and this control is an important element of gene regulation in both prokaryotic and eukaryotic cells . Of even broader significance, however, are the mechanisms that control the activities of proteins within cells. Once synthesized, most proteins can be regulated in response to extracellular signals by either covalent modifications or by association with other molecules. In addition, the levels of proteins within cells can be controlled by differential rates of protein degradation. These multiple controls of both the amounts and activities of intracellular proteins ultimately regulate all aspects of cell behavior.

• Translation of mRNA
• Protein Folding and Processing
• Regulation of Protein Function
• Protein Degradation
• References and Further Reading
• Cite this Page Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Chapter 7, Protein Synthesis, Processing, and Regulation.
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15.5 Ribosomes and Protein Synthesis

Learning objectives.

In this section, you will explore the following questions:

• What are the different sequential steps in protein synthesis?
• What is the role of ribosomes in protein synthesis?

Connection for AP ® Courses

After the information in the gene has been transcribed to mRNA, it is ready to be translated to polypeptide. The players in translation include the mRNA template, ribosomes, tRNA molecules, amino acids, and various enzymes. Ribosomes consist of small and large subunits of protein and rRNA which bind with mRNA; many ribosomes can move along the same mRNA at a time. Translation begins at the initiating AUG on mRNA, specifying methionine, the first amino acid in any polypeptide. Each amino acid is carried to the ribosome by attaching to a specific molecule of tRNA. A tRNA molecule often is depicted as a cloverleaf, with an anticodon on one end, and the amino acid attachment site at the other. Amino-acid charging enzymes ensure that the correct amino acid is attached to the correct tRNA. The anticodons on tRNA are complementary to the codons on mRNA; for example, the anticodon AAA on tRNA corresponds to TTT on mRNA. Sequential amino acids are linked by peptide bonds. The mRNA is translated, elongating the polypeptide, until a STOP or nonsense codon is reached. When this happens, a release factor dissociates the components and frees the new polypeptide. Folding of the protein occurs during and after translation. Once a polypeptide is synthesized, its role as a protein is established, such as determining a physical phenotype of an organism.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP ® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® Exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.

Teacher Support

Create models of protein synthesis with the following items:

• Pipe cleaners for RNA linking several units to represent mRNA and twisting some to represent tRNAs
• Cotton puff balls or other supplies to represent ribosomes
• Colored beads to represent amino acids and nucleotides

Ask students what specific challenges must face amino-acyl tRNA synthetases. The enzymes must recognize the anticodon, the amino acid that matches that anticodon, and the tRNA acceptor site.

Ask students to compare and contrast TATA boxes and Kozak’s sequences. Both are based on consensus sequences. TATA boxes are associated with promoters and Kozak’s sequences with binding of the ribosomes.

The RNA in the ribosomes catalyze the formation of the peptide bond. This is a good example of a ribozyme, an RNA molecule that acts as an enzyme. Students may have heard that all enzymes are proteins. This is an opportunity to clarify the point. The enzyme involved in the splicing of introns is another example of RNA with catalytic properties.

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards: [APLO 1.16][APLO 4.22][APLO 3.6]

The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform virtually every function of a cell. The process of translation, or protein synthesis, involves the decoding of an mRNA message into a polypeptide product. Amino acids are covalently strung together by interlinking peptide bonds in lengths ranging from approximately 50 amino acid residues to more than 1,000. Each individual amino acid has an amino group (NH 2 ) and a carboxyl (COOH) group. Polypeptides are formed when the amino group of one amino acid forms an amide (i.e., peptide) bond with the carboxyl group of another amino acid ( Figure 15.16 ). This reaction is catalyzed by ribosomes and generates one water molecule.

The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component may vary across species; for instance, ribosomes may consist of different numbers of rRNAs and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.

Link to Learning

Click through the steps of this PBS interactive to see protein synthesis in action.

• Due to lack of protein in the diet, our body will not be able to form other proteins; thus, it will conserve the protein it has for critical use, leading to hair loss.
• Lack of protein in the diet can weaken the immune system, thus leading to hair loss.
• Due to lack of protein in the diet, energy will be lost, thus leading to hair loss.
• Lack of protein in the diet will lead to breakage of disulfide bonds between proteins, thus leading to hair loss.

Even before an mRNA is translated, a cell must invest energy to build each of its ribosomes. In E. coli , there are between 10,000 and 70,000 ribosomes present in each cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.

Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and rough endoplasmic reticulum in eukaryotes. Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the ribosomes just outside their outer membranes in the cytoplasm. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli , the small subunit is described as 30S, and the large subunit is 50S, for a total of 70S (recall that Svedberg units are not additive). Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5' to 3' and synthesizing the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome .

The tRNAs are structural RNA molecules that were transcribed from genes by RNA polymerase III. Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins.

Of the 64 possible mRNA codons—or triplet combinations of A, U, G, and C—three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain. Of these 61, one codon (AUG) also encodes the initiation of translation. Each tRNA anticodon can base pair with one of the mRNA codons and add an amino acid or terminate translation, according to the genetic code. For instance, if the sequence CUA occurred on an mRNA template in the proper reading frame, it would bind a tRNA expressing the complementary sequence, GAU, which would be linked to the amino acid leucine.

As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. Consider that tRNAs need to interact with three factors: 1) they must be recognized by the correct aminoacyl synthetase (see below); 2) they must be recognized by ribosomes; and 3) they must bind to the correct sequence in mRNA.

Aminoacyl tRNA Synthetases

The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule. The corresponding amino acid must be added later, once the tRNA is processed and exported to the cytoplasm. Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by a group of enzymes called aminoacyl tRNA synthetases . At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between an amino acid and adenosine monophosphate (AMP); a pyrophosphate molecule is expelled in this reaction. The activated amino acid is then transferred to the tRNA, and AMP is released.

The Mechanism of Protein Synthesis

As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we’ll explore how translation occurs in E. coli , a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

Initiation of Translation

Protein synthesis begins with the formation of an initiation complex. In E. coli , this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special initiator tRNA , called t R N A f M e t t R N A f M e t . The initiator tRNA interacts with the start codon AUG (or rarely, GUG), links to a formylated methionine called fMet, and can also bind IF-2. Formylated methionine is inserted by f M e t − t R N A f M e t f M e t − t R N A f M e t at the beginning of every polypeptide chain synthesized by E. coli , but it is usually clipped off after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNA Met .

In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome’s translocation.

In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, IFs, and nucleoside triphosphates (GTP and ATP). The charged initiator tRNA, called Met-tRNA i , does not bind fMet in eukaryotes, but is distinct from other Met-tRNAs in that it can bind IFs.

Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex tracks along the mRNA in the 5' to 3' direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak’s rules , the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-gccRccAUGG-3'. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNA i , mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.

Translation, Elongation, and Termination

In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli . The 50S ribosomal subunit of E. coli consists of three compartments: the A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one exception to this assembly line of tRNAs: in E. coli , f M e t − t R N A f M e t f M e t − t R N A f M e t is capable of entering the P site directly without first entering the A site. Similarly, the eukaryotic Met-tRNA i , with help from other proteins of the initiation complex, binds directly to the P site. In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically.

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3' direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase , an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled ( Figure 15.17 ). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid protein can be translated in just 10 seconds.

Visual Connection

• tRNA binding to the ribosome
• Ribosome assembly
• Growth of the protein chain

Chloramphenicol would directly affect:

• Tetracycline would directly affect tRNA binding to the ribosome. Chloramphenicol would affect the growth of the protein chain.
• Tetracycline would directly affect ribosome assembly. Chloramphenicol would affect the growth of the protein chain.
• Tetracycline would directly affect the growth of the protein chain. Chloramphenicol would affect the tRNA binding to the ribosome.
• Tetracycline would directly affect mRNA binding to the ribosome. Chloramphenicol would affect the ribosome assembly.

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Protein Folding, Modification, and Targeting

During and after translation, individual amino acids may be chemically modified, signal sequences may be appended, and the new protein “folds” into a distinct three-dimensional structure as a result of intramolecular interactions. A signal sequence is a short tail of amino acids that directs a protein to a specific cellular compartment. These sequences at the amino end or the carboxyl end of the protein can be thought of as the protein’s “train ticket” to its ultimate destination. Other cellular factors recognize each signal sequence and help transport the protein from the cytoplasm to its correct compartment. For instance, a specific sequence at the amino terminus will direct a protein to the mitochondria or chloroplasts (in plants). Once the protein reaches its cellular destination, the signal sequence is usually clipped off.

Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding. Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape if abnormal temperature or pH conditions prevent it from folding correctly.

Science Practice Connection for AP® Courses

• Working in a small group, create a simple board game to model the key steps of transcription and translation and have classmates spend ten minutes playing the game.
• Provided with incomplete or incorrect diagrams illustrating transcription and translation in prokaryotes, have students refine or revise the diagrams and share the edited versions with classmates for critical review.

Think About It

• Many antibiotics inhibit protein synthesis. For example, tetracycline blocks the A site on the ribosome. What is the likely effect of tetracycline on protein synthesis?
• Using a chart of codons, transcribe and translate the following DNA sequence (non-template strand): 5′-ATGGCCGGTTATTAAGCA-3′. How can a single nucleotide change affect the protein produced from this sequence and its function?

The activities are applications of Learning Objective 3.4 and Science Practice 1.2 because students model how genetic information in DNA is ultimately translated into protein.

The first question is an application of Learning Objective 3.4 and Science Practice 1.2 because students are modeling how genetic information in DNA is ultimately translated into protein.

The second question is an application of Learning Objective 3.6 and Science Practice 6.4 because provided with a DNA sequence, students are asked to transcribe and translate the sequence and make a prediction about the possible effect of a mutation on the protein produced.

• Tetracycline would directly affect tRNA binding to the ribosome.
• The mRNA would be: 5'-AUGGCCGGUUAUUAAGCA-3'. The protein would be: MAGY (methionine-alanine-glycine-tyrosine.) Even though there are six codons, the fifth codon corresponds to a stop, so the sixth codon would not be translated. Responses to the second part of the inquiry may vary, as it would be dependent upon which nucleotide was changed. For example, if the A in the first codon (AUG) was changed to C, no protein would result.

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19 3.4 Protein Synthesis

Learning objectives.

By the end of this section, you will be able to:

• Explain how the genetic code stored within DNA determines the protein that will form
• Describe the process of transcription
• Describe the process of translation
• Discuss the function of ribosomes

It was mentioned earlier that DNA provides a “blueprint” for the cell structure and physiology. This refers to the fact that DNA contains the information necessary for the cell to build one very important type of molecule: the protein. Most structural components of the cell are made up, at least in part, by proteins and virtually all the functions that a cell carries out are completed with the help of proteins. One of the most important classes of proteins is enzymes, which help speed up necessary biochemical reactions that take place inside the cell. Some of these critical biochemical reactions include building larger molecules from smaller components (such as occurs during DNA replication or synthesis of microtubules) and breaking down larger molecules into smaller components (such as when harvesting chemical energy from nutrient molecules). Whatever the cellular process may be, it is almost sure to involve proteins. Just as the cell’s genome describes its full complement of DNA, a cell’s proteome is its full complement of proteins. Protein synthesis begins with genes. A gene is a functional segment of DNA that provides the genetic information necessary to build a protein. Each particular gene provides the code necessary to construct a particular protein. Gene expression , which transforms the information coded in a gene to a final gene product, ultimately dictates the structure and function of a cell by determining which proteins are made.

The interpretation of genes works in the following way. Recall that proteins are polymers, or chains, of many amino acid building blocks. The sequence of bases in a gene (that is, its sequence of A, T, C, G nucleotides) translates to an amino acid sequence. A triplet is a section of three DNA bases in a row that codes for a specific amino acid. Similar to the way in which the three-letter code d-o-g signals the image of a dog, the three-letter DNA base code signals the use of a particular amino acid. For example, the DNA triplet CAC (cytosine, adenine, and cytosine) specifies the amino acid valine. Therefore, a gene, which is composed of multiple triplets in a unique sequence, provides the code to build an entire protein, with multiple amino acids in the proper sequence ( Figure 1 ). The mechanism by which cells turn the DNA code into a protein product is a two-step process, with an RNA molecule as the intermediate.

From DNA to RNA: Transcription

DNA is housed within the nucleus, and protein synthesis takes place in the cytoplasm, thus there must be some sort of intermediate messenger that leaves the nucleus and manages protein synthesis. This intermediate messenger is messenger RNA (mRNA) , a single-stranded nucleic acid that carries a copy of the genetic code for a single gene out of the nucleus and into the cytoplasm where it is used to produce proteins.

There are several different types of RNA, each having different functions in the cell. The structure of RNA is similar to DNA with a few small exceptions. For one thing, unlike DNA, most types of RNA, including mRNA, are single-stranded and contain no complementary strand. Second, the ribose sugar in RNA contains an additional oxygen atom compared with DNA. Finally, instead of the base thymine, RNA contains the base uracil. This means that adenine will always pair up with uracil during the protein synthesis process.

Gene expression begins with the process called transcription , which is the synthesis of a strand of mRNA that is complementary to the gene of interest. This process is called transcription because the mRNA is like a transcript, or copy, of the gene’s DNA code. Transcription begins in a fashion somewhat like DNA replication, in that a region of DNA unwinds and the two strands separate, however, only that small portion of the DNA will be split apart. The triplets within the gene on this section of the DNA molecule are used as the template to transcribe the complementary strand of RNA ( Figure 2 ). A codon is a three-base sequence of mRNA, so-called because they directly encode amino acids. Like DNA replication, there are three stages to transcription: initiation, elongation, and termination.

Stage 1: Initiation. A region at the beginning of the gene called a promoter —a particular sequence of nucleotides—triggers the start of transcription.

Stage 2: Elongation. Transcription starts when RNA polymerase unwinds the DNA segment. One strand, referred to as the coding strand, becomes the template with the genes to be coded. The polymerase then aligns the correct nucleic acid (A, C, G, or U) with its complementary base on the coding strand of DNA. RNA polymerase is an enzyme that adds new nucleotides to a growing strand of RNA. This process builds a strand of mRNA.

Stage 3: Termination. When the polymerase has reached the end of the gene, one of three specific triplets (UAA, UAG, or UGA) codes a “stop” signal, which triggers the enzymes to terminate transcription and release the mRNA transcript.

Before the mRNA molecule leaves the nucleus and proceeds to protein synthesis, it is modified in a number of ways. For this reason, it is often called a pre-mRNA at this stage. For example, your DNA, and thus complementary mRNA, contains long regions called non-coding regions that do not code for amino acids. Their function is still a mystery, but the process called splicing removes these non-coding regions from the pre-mRNA transcript ( Figure 3 ). A spliceosome —a structure composed of various proteins and other molecules—attaches to the mRNA and “splices” or cuts out the non-coding regions. The removed segment of the transcript is called an intron . The remaining exons are pasted together. An exon is a segment of RNA that remains after splicing. Interestingly, some introns that are removed from mRNA are not always non-coding. When different coding regions of mRNA are spliced out, different variations of the protein will eventually result, with differences in structure and function. This process results in a much larger variety of possible proteins and protein functions. When the mRNA transcript is ready, it travels out of the nucleus and into the cytoplasm.

From RNA to Protein: Translation

Like translating a book from one language into another, the codons on a strand of mRNA must be translated into the amino acid alphabet of proteins. Translation is the process of synthesizing a chain of amino acids called a polypeptide . Translation requires two major aids: first, a “translator,” the molecule that will conduct the translation, and second, a substrate on which the mRNA strand is translated into a new protein, like the translator’s “desk.” Both of these requirements are fulfilled by other types of RNA. The substrate on which translation takes place is the ribosome.

Remember that many of a cell’s ribosomes are found associated with the rough ER, and carry out the synthesis of proteins destined for the Golgi apparatus. Ribosomal RNA (rRNA) is a type of RNA that, together with proteins, composes the structure of the ribosome. Ribosomes exist in the cytoplasm as two distinct components, a small and a large subunit. When an mRNA molecule is ready to be translated, the two subunits come together and attach to the mRNA. The ribosome provides a substrate for translation, bringing together and aligning the mRNA molecule with the molecular “translators” that must decipher its code.

The other major requirement for protein synthesis is the translator molecules that physically “read” the mRNA codons. Transfer RNA (tRNA) is a type of RNA that ferries the appropriate corresponding amino acids to the ribosome, and attaches each new amino acid to the last, building the polypeptide chain one-by-one. Thus tRNA transfers specific amino acids from the cytoplasm to a growing polypeptide. The tRNA molecules must be able to recognize the codons on mRNA and match them with the correct amino acid. The tRNA is modified for this function. On one end of its structure is a binding site for a specific amino acid. On the other end is a base sequence that matches the codon specifying its particular amino acid. This sequence of three bases on the tRNA molecule is called an anticodon . For example, a tRNA responsible for shuttling the amino acid glycine contains a binding site for glycine on one end. On the other end it contains an anticodon that complements the glycine codon (GGA is a codon for glycine, and so the tRNAs anticodon would read CCU). Equipped with its particular cargo and matching anticodon, a tRNA molecule can read its recognized mRNA codon and bring the corresponding amino acid to the growing chain ( Figure 4 ).

Much like the processes of DNA replication and transcription, translation consists of three main stages: initiation, elongation, and termination. Initiation takes place with the binding of a ribosome to an mRNA transcript. The elongation stage involves the recognition of a tRNA anticodon with the next mRNA codon in the sequence. Once the anticodon and codon sequences are bound (remember, they are complementary base pairs), the tRNA presents its amino acid cargo and the growing polypeptide strand is attached to this next amino acid. This attachment takes place with the assistance of various enzymes and requires energy. The tRNA molecule then releases the mRNA strand, the mRNA strand shifts one codon over in the ribosome, and the next appropriate tRNA arrives with its matching anticodon. This process continues until the final codon on the mRNA is reached which provides a “stop” message that signals termination of translation and triggers the release of the complete, newly synthesized protein. Thus, a gene within the DNA molecule is transcribed into mRNA, which is then translated into a protein product ( Figure 5 ).

Commonly, an mRNA transcription will be translated simultaneously by several adjacent ribosomes. This increases the efficiency of protein synthesis. A single ribosome might translate an mRNA molecule in approximately one minute; so multiple ribosomes aboard a single transcript could produce multiple times the number of the same protein in the same minute. A polyribosome is a string of ribosomes translating a single mRNA strand.

Watch this video to learn about ribosomes. The ribosome binds to the mRNA molecule to start translation of its code into a protein. What happens to the small and large ribosomal subunits at the end of translation?

Chapter Review

DNA stores the information necessary for instructing the cell to perform all of its functions. Cells use the genetic code stored within DNA to build proteins, which ultimately determine the structure and function of the cell. This genetic code lies in the particular sequence of nucleotides that make up each gene along the DNA molecule. To “read” this code, the cell must perform two sequential steps. In the first step, transcription, the DNA code is converted into a RNA code. A molecule of messenger RNA that is complementary to a specific gene is synthesized in a process similar to DNA replication. The molecule of mRNA provides the code to synthesize a protein. In the process of translation, the mRNA attaches to a ribosome. Next, tRNA molecules shuttle the appropriate amino acids to the ribosome, one-by-one, coded by sequential triplet codons on the mRNA, until the protein is fully synthesized. When completed, the mRNA detaches from the ribosome, and the protein is released. Typically, multiple ribosomes attach to a single mRNA molecule at once such that multiple proteins can be manufactured from the mRNA concurrently.

Interactive Link Questions

They separate and move and are free to join translation of other segments of mRNA.

Review Questions

1. Which of the following is not a difference between DNA and RNA?

• DNA contains thymine whereas RNA contains uracil
• DNA contains deoxyribose and RNA contains ribose
• DNA contains alternating sugar-phosphate molecules whereas RNA does not contain sugars
• RNA is single stranded and DNA is double stranded

2. Transcription and translation take place in the ________ and ________, respectively.

• nucleus; cytoplasm
• nucleolus; nucleus
• nucleolus; cytoplasm
• cytoplasm; nucleus

3. How many “letters” of an RNA molecule, in sequence, does it take to provide the code for a single amino acid?

4. Which of the following is not made out of RNA?

• the carriers that shuffle amino acids to a growing polypeptide strand
• the ribosome
• the messenger molecule that provides the code for protein synthesis

Critical Thinking Questions

1. Briefly explain the similarities between transcription and DNA replication.

2.Contrast transcription and translation. Name at least three differences between the two processes.

Answers for Review Questions

Answers for Critical Thinking Questions

• Transcription and DNA replication both involve the synthesis of nucleic acids. These processes share many common features—particularly, the similar processes of initiation, elongation, and termination. In both cases the DNA molecule must be untwisted and separated, and the coding (i.e., sense) strand will be used as a template. Also, polymerases serve to add nucleotides to the growing DNA or mRNA strand. Both processes are signaled to terminate when completed.
• Transcription is really a “copy” process and translation is really an “interpretation” process, because transcription involves copying the DNA message into a very similar RNA message whereas translation involves converting the RNA message into the very different amino acid message. The two processes also differ in their location: transcription occurs in the nucleus and translation in the cytoplasm. The mechanisms by which the two processes are performed are also completely different: transcription utilizes polymerase enzymes to build mRNA whereas translation utilizes different kinds of RNA to build protein.

Anatomy and Physiology Copyright © 1999-2016 by Rice University is licensed under a Creative Commons Attribution 4.0 International License , except where otherwise noted.

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25.13: Biosynthesis of Proteins

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• Page ID 22368

• John D. Roberts and Marjorie C. Caserio
• California Institute of Technology

One of the most interesting and basic problems connected with the synthesis of proteins in living cells is how the component amino acids are induced to link together in the sequences that are specific for each type of protein. There also is the related problem of how the information as to the amino-acid sequences is perpetuated in each new generation of cells. We now know that the substances responsible for genetic control in plants and animals are present in and originate from the chromosomes of cell nuclei. Chemical analysis of the chromosomes has revealed them to be composed of giant molecules of deoxyribonucleoproteins, which are deoxyribonucleic acids (DNA) bonded to proteins. Since it is known that DNA rather than the protein component of a nucleoprotein contains the genetic information for the biosynthesis of enzymes and other proteins, we shall be interested mainly in DNA and will first discuss its structure. Part or perhaps all of a particular DNA is the chemical equivalent of the Mendelian gene - the unit of inheritance.

The Structure of DNA

The role of DNA in living cells is analogous to that of a punched tape used for controlling the operation of an automatic turret lathe - DNA supplies the information for the development of the cells, including synthesis of the necessary enzymes and such replicas of itself as are required for reproduction by cell division. Obviously, we would not expect the DNA of one kind of organism to be the same as DNA of another kind of organism. It is therefore impossible to be very specific about the structure of DNA without being specific about the organism from which it is derived. Nonetheless, the basic structural features of DNA are the same for many kinds of cells, and we mainly shall be concerned with these basic features in the following discussion.

In the first place, DNA molecules are quite large, sufficiently so to permit them to be seen individually in photographs taken with electron microscopes. The molecular weights vary considerably, but values of 1,000,000 to 4,000,000,000 are typical. X-ray diffraction indicates that DNA is made up of two long-chain molecules twisted around each other to form a double-stranded helix about \(20 \: \text{Å}\) in diameter. The arrangement is shown schematically in \(12\):

As we shall see, the components of the chains are such that the strands can be held together efficiently by hydrogen bonds. In agreement with this structure, it has been found that, when DNA is heated to about \(80^\text{o}\) under proper conditions, the strands of the helix unwind and dissociate into two randomly coiled fragments. Furthermore, when the dissociated material is allowed to cool slowly under the proper conditions, the fragments recombine and regenerate the helical structure.

Chemical studies show that the strands of DNA have the structure of a long-chain polymer made of alternating phosphate and sugar residues carrying nitrogen bases, \(13\):

The sugar is \(D\)-2-deoxyribofuranose, \(14\), and each sugar residue is bonded to two phosphate groups by way of ester links involving the 3- and 5-hydroxyl groups:

The backbone of DNA is thus a polyphosphate ester of a 1,3-diol:

Each of the sugar residues of DNA is bonded at the 1-position of one of four bases: cytosine, \(15\); thymine, \(16\); adenine, \(17\); and guanine, \(18\). The four bases are derivatives of either pyrimidine or purine , both of which are heterocyclic nitrogen bases:

Unlike phenols ( Section 26-1 ), structural analysis of many of the hydroxy-substituted aza-aromatic compounds is complicated by isomerism of the keto-enol type, sometimes called lactim-lactam isomerism. For 2-hydroxypyrimidine, \(19\), these isomers are \(19a\) and \(19b\), and the lactam form is more stable, as also is true for cytosine, \(15\), thymine, \(16\), and the pyrimidine ring of guanine, \(18\).

For the sake of simplicity in illustrating \(\ce{N}\)-glycoside formation in DNA, we shall show the type of bonding involved for the sugar and base components only (i.e., the deoxyribose nucleoside structure). Attachment of 2-deoxyribose is through a \(\ce{NH}\) group to form the \(\beta\)-\(\ce{N}\)-deoxyribofuranoside ( Section 20-5 ):

Esterification of the 5'-hydroxyl group of deoxyribose nucleosides , such as cytosine deoxyriboside, with phosphoric acid gives the corresponding nucleotides :\(^{11}\)

The number of nucleotide units in a DNA chain varies from about 3,000 to 10,000,000 Although the sequence of the purine and pyrimidine bases in the chains are not known, there is a striking equivalence between the numbers of certain of the bases regardless of the origin of DNA. Thus the number of adenine (A) groups equals the number of thymine (T) groups, and the number of guanine (G) groups equals the number of cytosine (C) groups: A = T and G = C. The bases of DNA therefore are half purines and half pyrimidines. Furthermore, although the ratios of A to G and T to C are constant for a given species, they vary widely from one species to another.

The equivalence between the purine and pyrimidine bases in DNA was accounted for by J. D. Watson and F. Crick (1953) through the suggestion that the two strands are constructed so that, when twisted together in the helical structure, hydrogen bonds are formed involving adenine in one chain and thymine in the other, or cytosine in one chain and guanine in the other. Thus each adenine occurs paired with a thymine and each cytosine with a guanine and the strands are said to have complementary structures. The postulated hydrogen bonds are shown in Figure 25-22, and the relationship of the bases to the strands in Figure 25-23.

Genetic Control and the Replication of DNA

It is now well established that DNA provides the genetic recipe that determines how cells reproduce. In the process of cell division, the DNA itself also is reproduced and thus perpetuates the information necessary to regulate the synthesis of specific enzymes and other proteins of the cell structure. In replicating itself prior to cell division, the DNA double helix evidently separates at least partly into two strands (see Figure 25-24). Each of the separated parts serves as a guide (template) for the assembly of a complementary sequence of nucleotides along its length. Ultimately, two new DNA double strands are formed, each of which contains one strand from the parent DNA.

The genetic information inherent in DNA depends on the arrangement of the bases (A, T, G, and C) along the phosphate-carbohydrate backbone - that is, on the arrangement of the four nucleotides specific to DNA. Thus the sequence A-G-C at a particular point conveys a different message than the sequence G-A-C.

It is quite certain that the code involves a particular sequence of three nucleotides for each amino acid. Thus the sequence A-A-A codes for lysine, and U-C-G codes for serine. The sequences or codons for all twenty amino acids are known.

Role of RNA in Synthesis of Proteins

It is clear that DNA does not play a direct role in the synthesis of proteins and enzymes because most of the protein synthesis takes place outside of the cell nucleus in the cellular cytoplasm, which does not contain DNA. Furthermore, it has been shown that protein synthesis can occur in the absence of a cell nucleus or, equally, in the absence of DNA. Therefore the genetic code in DNA must be passed on selectively to other substances that carry information from the nucleus to the sites of protein synthesis in the cytoplasm. These other substances are ribonucleic acids (RNA), which are polymeric molecules similar in structure to DNA, except that \(D\)-2-deoxyribofuranose is replaced by \(D\)-ribofuranose and the base thymine is replace by uracil, as shown in Figure 25-25.

RNA also differs from DNA in that there are not the same regularities in the overall composition of its bases and it usually consists of a single polynucleotide chain. There are different types of RNA, which fulfill different functions. About \(80\%\) of the RNA in a cell is located in the cytoplasm in clusters closely associated with proteins. These ribonucleoprotein particles specifically are called ribosomes, and the ribosomes are the sites of most of the protein synthesis in the cell. In addition to the ribosomal RNA (rRNA), there are ribonucleic acids called messenger RNA (mRNA), which convey instructions as to what protein to make. In addition, there are ribonucleic acids called transfer RNA (tRNA), which actually guide the amino acids into place in protein synthesis. Much is now known about the structure and function of tRNA.

The principal structural features of tRNA molecules are shown schematically in Figure 25-26. Some of the important characteristics of tRNA molecules are summarized as follows.

1. There is at least one particular tRNA for each amino acid. 2. The tRNA molecules have single chains with 73-93 ribonucleotides. Most of the tRNA bases are adenine (A), cytosine (C), guanine (G), and uracil (U). There also are a number of unusual bases that are methylated derivatives of A, C, G, and U. 3. The clover-leaf pattern of Figure 25-26 shows the general structure of tRNA. There are regions of the chain where the bases are complementary to one another, which causes it to fold into two double-helical regions. The chain has three bends or loops separating the helical regions. 4. The 5'-terminal residue usually is a guanine nucleotide; it is phosphorylated at the 5'-\(\ce{OH}\). The terminus at the 3' end has the same sequence of three nucleotides in all tRNA's, namely, CCA. The 3'-\(\ce{OH}\) of the adenosine in this grouping is the point of attachment of the tRNA to its specific amino acid:

With this information on the structure of tRNA, we can proceed to a discussion of the essential features of biochemical protein synthesis.

The information that determines amino-acid sequence in a protein to be synthesized is contained in the DNA of a cell nucleus as a particular sequence of nucleotides derived from adenine, guanine, thymine, and cytosine. For each particular amino acid there is a sequence of three nucleotides called a codon .

The information on protein structure is transmitted from the DNA in the cell nucleus to the cytoplasm where the protein is assembled by messenger RNA. This messenger RNA, or at least part of it, is assembled in the nucleus with a base sequence that is complementary to the base sequence in the parent DNA. The assembly mechanism is similar to DNA replication except that thymine (T) is replaced by uracil (U). The uracil is complementary to adenine in the DNA chain (see Figure 25-27). After the mRNA is assembled, it is transported to the cytoplasm where it becomes attached to the ribosomes.

The amino acids in the cytoplasm will not form polypeptides unless activated by ester formation with appropriate tRNA molecules. The ester linkages are through the 3'-\(\ce{OH}\) of the terminal adenosine nucleotide (Equation 25-9) and are formed only under the influence of a synthetase enzyme that is specific for the particular amino acid. The energy for ester formation comes from ATP hydrolysis ( Sections 15-5F and 20-10 ). The product is called an aminoacyl-tRNA .

The aminoacyl-tRNA's form polypeptide chains in the order specified by codons of the mRNA bound to the ribosomes (see Figure 25-28). The order of incorporation of the amino acids depends on the recognition of a codon in mRNA by the corresponding anticodon in tRNA by a complementary base-pairing of the type A \(\cdots\) U and C \(\cdots\) G. The first two bases of the codon recognize only their complementary bases in the anticodon, but there is some flexibility in the identity of the third base. Thus phenylalanine tRNA has the anticodon A-A-G and responds to the codons U-U-C and U-U-U, but not U-U-A or U-U-G:

The codons of the mRNA on the ribosomes are read from the 5' to the 3' end. Thus the synthetic polynucleotide (5')A-A-A-(A-A-A)\(_n\)-A-A-C(3') contains the code for lysine (A-A-A) and asparagine (A-A-C); the actual polypeptide obtained using this mRNA in a cell-free system was Lys-(Lys)\(_n\)-Asn, and not Asn-(Lys)\(_n\)-Lys.

The start of protein synthesis is signaled by specific codon-anticodon interactions. Termination is also signaled by a codon in the mRNA, although the stop signal is not recognized by tRNA, but by proteins that then trigger the hydrolysis of the completed polypeptide chain from the tRNA. Just how the secondary and tertiary structures of the proteins are achieved is not yet clear, but certainly the mechanism of protein synthesis, which we have outlined here, requires little modification to account for preferential formation of particular conformations.

\(^{11}\)The positions on the sugar ring are primed to differentiate them from the positions of the nitrogen base.

Contributors and Attributions

John D. Robert and Marjorie C. Caserio (1977) Basic Principles of Organic Chemistry, second edition. W. A. Benjamin, Inc. , Menlo Park, CA. ISBN 0-8053-8329-8. This content is copyrighted under the following conditions, "You are granted permission for individual, educational, research and non-commercial reproduction, distribution, display and performance of this work in any format."

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2.9 Protein Synthesis

Learning Objectives

By the end of this section, you will be able to:

• Explain how the genetic code stored within DNA determines the protein that will form
• Describe the process of transcription
• Describe the process of translation
• Discuss the function of ribosomes

It was mentioned earlier that DNA provides a “blueprint” for the cell structure and physiology. This refers to the fact that DNA contains the information necessary for the cell to build one very important type of molecule: the protein. Most structural components of the cell are made up, at least in part, by proteins and virtually all the functions that a cell carries out are completed with the help of proteins. One of the most important classes of proteins is enzymes, which help speed up necessary biochemical reactions that take place inside the cell. Some of these critical biochemical reactions include building larger molecules from smaller components (such as occurs during DNA replication or synthesis of microtubules) and breaking down larger molecules into smaller components (such as when harvesting chemical energy from nutrient molecules). Whatever the cellular process may be, it is almost sure to involve proteins. Just as the cell’s genome describes its full complement of DNA, a cell’s  proteome  is its full complement of proteins. Protein synthesis begins with genes. A  gene  is a functional segment of DNA that provides the genetic information necessary to build a protein. Each particular gene provides the code necessary to construct a particular protein.  Gene expression , which transforms the information coded in a gene to a final gene product, ultimately dictates the structure and function of a cell by determining which proteins are made.

The interpretation of genes works in the following way. Recall that proteins are polymers, or chains, of many amino acid building blocks. The sequence of bases in a gene (that is, its sequence of A, T, C, G nucleotides) translates to an amino acid sequence. A  triplet  is a section of three DNA bases in a row that codes for a specific amino acid. Similar to the way in which the three-letter code  d-o-g  signals the image of a dog, the three-letter DNA base code signals the use of a particular amino acid. For example, the DNA triplet CAC (cytosine, adenine, and cytosine) specifies the amino acid valine. Therefore, a gene, which is composed of multiple triplets in a unique sequence, provides the code to build an entire protein, with multiple amino acids in the proper sequence (Figure 2.9.1). The mechanism by which cells turn the DNA code into a protein product is a two-step process, with an RNA molecule as the intermediate.

From DNA to RNA: Transcription

DNA is housed within the nucleus, and protein synthesis takes place in the cytoplasm, thus there must be some sort of intermediate messenger that leaves the nucleus and manages protein synthesis. This intermediate messenger is  messenger RNA (mRNA) , a single-stranded nucleic acid that carries a copy of the genetic code for a single gene out of the nucleus and into the cytoplasm where it is used to produce proteins.

There are several different types of RNA, each having different functions in the cell. The structure of RNA is similar to DNA with a few small exceptions. For one thing, unlike DNA, most types of RNA, including mRNA, are single-stranded and contain no complementary strand. Second, the ribose sugar in RNA contains an additional oxygen atom compared with DNA. Finally, instead of the base thymine, RNA contains the base uracil. This means that adenine will always pair up with uracil during the protein synthesis process.

Gene expression begins with the process called  transcription , which is the synthesis of a strand of mRNA that is complementary to the gene of interest. This process is called transcription because the mRNA is like a transcript, or copy, of the gene’s DNA code. Transcription begins in a fashion somewhat like DNA replication, in that a region of DNA unwinds and the two strands separate, however, only that small portion of the DNA will be split apart. The triplets within the gene on this section of the DNA molecule are used as the template to transcribe the complementary strand of RNA (Figure 2.9.2). A  codon  is a three-base sequence of mRNA, so-called because they directly encode amino acids. Like DNA replication, there are three stages to transcription: initiation, elongation, and termination.

Stage 1: Initiation .  A region at the beginning of the gene called a  promoter —a particular sequence of nucleotides—triggers the start of transcription.

Stage 2: Elongation .  Transcription starts when RNA polymerase unwinds the DNA segment. One strand, referred to as the coding strand, becomes the template with the genes to be coded. The polymerase then aligns the correct nucleic acid (A, C, G, or U) with its complementary base on the coding strand of DNA.  RNA polymerase  is an enzyme that adds new nucleotides to a growing strand of RNA. This process builds a strand of mRNA.

Stage 3: Termination .  When the polymerase has reached the end of the gene, one of three specific triplets (UAA, UAG, or UGA) codes a “stop” signal, which triggers the enzymes to terminate transcription and release the mRNA transcript.

Before the mRNA molecule leaves the nucleus and proceeds to protein synthesis, it is modified in several ways. For this reason, it is often called a pre-mRNA at this stage. For example, your DNA, and thus complementary mRNA, contains long regions called non-coding regions that do not code for amino acids. Their function is still a mystery, but the process called  splicing  removes these non-coding regions from the pre-mRNA transcript (Figure 2.9.3). A  spliceosome —a structure composed of various proteins and other molecules—attaches to the mRNA and “splices” or cuts out the non-coding regions. The removed segment of the transcript is called an  intron . The remaining exons are pasted together. An  exon  is a segment of RNA that remains after splicing. Interestingly, some introns that are removed from mRNA are not always non-coding. When different coding regions of mRNA are spliced out, different variations of the protein will eventually result, with differences in structure and function. This process results in a much larger variety of possible proteins and protein functions. When the mRNA transcript is ready, it travels out of the nucleus and into the cytoplasm.

From RNA to Protein: Translation

Like translating a book from one language into another, the codons on a strand of mRNA must be translated into the amino acid alphabet of proteins.  Translation  is the process of synthesising a chain of amino acids called a  polypeptide . Translation requires two major aids: first, a “translator,” the molecule that will conduct the translation, and second, a substrate on which the mRNA strand is translated into a new protein, like the translator’s “desk.” Both of these requirements are fulfilled by other types of RNA. The substrate on which translation takes place is the ribosome.

Remember that many of a cell’s ribosomes are found associated with the rough ER, and carry out the synthesis of proteins destined for the Golgi apparatus.  Ribosomal RNA (rRNA)  is a type of RNA that, together with proteins, composes the structure of the ribosome. Ribosomes exist in the cytoplasm as two distinct components, a small and a large subunit. When an mRNA molecule is ready to be translated, the two subunits come together and attach to the mRNA. The ribosome provides a substrate for translation, bringing together and aligning the mRNA molecule with the molecular “translators” that must decipher its code.

The other major requirement for protein synthesis is the translator molecules that physically “read” the mRNA codons.  Transfer RNA (tRNA)  is a type of RNA that ferries the appropriate corresponding amino acids to the ribosome, and attaches each new amino acid to the last, building the polypeptide chain one-by-one. Thus, tRNA transfers specific amino acids from the cytoplasm to a growing polypeptide. The tRNA molecules must be able to recognise the codons on mRNA and match them with the correct amino acid. The tRNA is modified for this function. On one end of its structure is a binding site for a specific amino acid. On the other end is a base sequence that matches the codon specifying its particular amino acid. This sequence of three bases on the tRNA molecule is called an  anticodon . For example, a tRNA responsible for shuttling the amino acid glycine contains a binding site for glycine on one end. On the other end it contains an anticodon that complements the glycine codon (GGA is a codon for glycine, and so the tRNAs anticodon would read CCU). Equipped with its particular cargo and matching anticodon, a tRNA molecule can read its recognised mRNA codon and bring the corresponding amino acid to the growing chain (Figure 2.9.4).

Much like the processes of DNA replication and transcription, translation consists of three main stages: initiation, elongation, and termination. Initiation takes place with the binding of a ribosome to an mRNA transcript. The elongation stage involves the recognition of a tRNA anticodon with the next mRNA codon in the sequence. Once the anticodon and codon sequences are bound (remember, they are complementary base pairs), the tRNA presents its amino acid cargo and the growing polypeptide strand is attached to this next amino acid. This attachment takes place with the assistance of various enzymes and requires energy. The tRNA molecule then releases the mRNA strand, the mRNA strand shifts one codon over in the ribosome, and the next appropriate tRNA arrives with its matching anticodon. This process continues until the final codon on the mRNA is reached which provides a “stop” message that signals termination of translation and triggers the release of the complete, newly synthesised protein. Thus, a gene within the DNA molecule is transcribed into mRNA, which is then translated into a protein product (Figure 2.9.5).

Commonly, an mRNA transcription will be translated simultaneously by several adjacent ribosomes. This increases the efficiency of protein synthesis. A single ribosome might translate an mRNA molecule in approximately one minute; so multiple ribosomes aboard a single transcript could produce multiple times the number of the same protein in the same minute. A polyribosome is a string of ribosomes translating a single mRNA strand.

Section Review

DNA stores the information necessary for instructing the cell to perform all its functions. Cells use the genetic code stored within DNA to build proteins, which ultimately determine the structure and function of the cell. This genetic code lies in the particular sequence of nucleotides that make up each gene along the DNA molecule. To “read” this code, the cell must perform two sequential steps. In the first step, transcription, the DNA code is converted into a RNA code. A molecule of messenger RNA that is complementary to a specific gene is synthesised in a process like DNA replication. The molecule of mRNA provides the code to synthesise a protein. In the process of translation, the mRNA attaches to a ribosome. Next, tRNA molecules shuttle the appropriate amino acids to the ribosome, one-by-one, coded by sequential triplet codons on the mRNA, until the protein is fully synthesised. When completed, the mRNA detaches from the ribosome, and the protein is released. Typically, multiple ribosomes attach to a single mRNA molecule at once such that multiple proteins can be manufactured from the mRNA concurrently.

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5.4: Protein Synthesis (Translation)

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Learning Objectives

• Describe the genetic code and explain why it is considered almost universal
• Explain the process of translation and the functions of the molecular machinery of translation
• Compare translation in eukaryotes and prokaryotes

The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation, or protein synthesis, the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.

The Genetic Code

Translation of the mRNA template converts nucleotide-based genetic information into the “language” of amino acids to create a protein product. A protein sequence consists of 20 commonly occurring amino acids. Each amino acid is defined within the mRNA by a triplet of nucleotides called a codon. The relationship between an mRNA codon and its corresponding amino acid is called the genetic code.

The three-nucleotide code means that there is a total of 64 possible combinations (4 3 , with four different nucleotides possible at each of the three different positions within the codon). This number is greater than the number of amino acids and a given amino acid is encoded by more than one codon (Figure \(\PageIndex{1}\)). This redundancy in the genetic code is called degeneracy. Typically, whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing polypeptide, the third position, called the wobble position, is less critical. In some cases, if the nucleotide in the third position is changed, the same amino acid is still incorporated.

Whereas 61 of the 64 possible triplets code for amino acids, three of the 64 codons do not code for an amino acid; they terminate protein synthesis, releasing the polypeptide from the translation machinery. These are called stop codons or nonsense codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also typically serves as the start codon to initiate translation. The reading frame, the way nucleotides in mRNA are grouped into codons, for translation is set by the AUG start codon near the 5’ end of the mRNA. Each set of three nucleotides following this start codon is a codon in the mRNA message.The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all extant life on earth shares a common origin. However, unusual amino acids such as selenocysteine and pyrrolysine have been observed in archaea and bacteria. In the case of selenocysteine, the codon used is UGA (normally a stop codon). However, UGA can encode for selenocysteine using a stem-loop structure (known as the selenocysteine insertion sequence, or SECIS element), which is found at the 3’ untranslated region of the mRNA. Pyrrolysine uses a different stop codon, UAG. The incorporation of pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a CUA anticodon

Exercise \(\PageIndex{1}\)

• How many bases are in each codon?
• What amino acid is coded for by the codon AAU?
• What happens when a stop codon is reached?

The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component varies across taxa; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNAs) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.

A ribosome is a complex macromolecule composed of catalytic rRNAs (called ribozymes) and structural rRNAs, as well as many distinct polypeptides. Mature rRNAs make up approximately 50% of each ribosome. Prokaryotes have 70S ribosomes, whereas eukaryotes have 80S ribosomes in the cytoplasm and rough endoplasmic reticulum, and 70S ribosomes in mitochondria and chloroplasts. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli , the small subunit is described as 30S (which contains the 16S rRNA subunit), and the large subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of 70S (Svedberg units are not additive). Eukaryote ribosomes have a small 40S subunit (which contains the 18S rRNA subunit) and a large 60S subunit (which contains the 5S, 5.8S and 28S rRNA subunits), for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit binds tRNAs (discussed in the next subsection).

Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5’ to 3’ and synthesizing the polypeptide from the N terminus to the C terminus. The complete structure containing an mRNA with multiple associated ribosomes is called a polyribosome (or polysome). In both bacteria and archaea, before transcriptional termination occurs, each protein-encoding transcript is already being used to begin synthesis of numerous copies of the encoded polypeptide(s) because the processes of transcription and translation can occur concurrently, forming polyribosomes (Figure \(\PageIndex{2}\)). The reason why transcription and translation can occur simultaneously is because both of these processes occur in the same 5’ to 3’ direction, they both occur in the cytoplasm of the cell, and because the RNA transcript is not processed once it is transcribed. This allows a prokaryotic cell to respond to an environmental signal requiring new proteins very quickly. In contrast, in eukaryotic cells, simultaneous transcription and translation is not possible. Although polyribosomes also form in eukaryotes, they cannot do so until RNA synthesis is complete and the RNA molecule has been modified and transported out of the nucleus.

Transfer RNAs

Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA.

Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other (Figure \(\PageIndex{3}\)). This shape positions the amino-acid binding site, called the CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3’ end of the tRNA, and the anticodon at the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.

An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is released.

Exercise \(\PageIndex{2}\)

• Describe the structure and composition of the prokaryotic ribosome.
• In what direction is the mRNA template read?
• Describe the structure and function of a tRNA.

The Mechanism of Protein Synthesis

Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli , a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.

The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli , this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N -formyl-methionine(fMet-tRNA fMet ) (Figure \(\PageIndex{4}\)). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli . In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.

In eukaryotes, initiation complex formation is similar, with the following differences:

• The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
• Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5’ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5’ to 3’ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli , the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNA fMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex.

In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure \(\PageIndex{5}\) and listed in Figure \(\PageIndex{6}\).

Protein Targeting, Folding, and Modification

During and after translation, polypeptides may need to be modified before they are biologically active. Post-translational modifications include:

• removal of translated signal sequences—short tails of amino acids that aid in directing a protein to a specific cellular compartment
• proper “folding” of the polypeptide and association of multiple polypeptide subunits, often facilitated by chaperone proteins, into a distinct three-dimensional structure
• proteolytic processing of an inactive polypeptide to release an active protein component, and
• various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of individual amino acids.

Exercise \(\PageIndex{3}\)

• What are the components of the initiation complex for translation in prokaryotes?
• What are two differences between initiation of prokaryotic and eukaryotic translation?
• What occurs at each of the three active sites of the ribosome?
• What causes termination of translation?

Key Concepts and Summary

• In translation , polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match mRNA codons to specific amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.
• The genetic code is degenerate in that several mRNA codons code for the same amino acids. The genetic code is almost universal among living organisms.
• Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large subunit and a small subunit of differing sizes between the two groups. Each subunit is composed of rRNA and protein. Organelle ribosomes in eukaryotic cells resemble prokaryotic ribosomes.
• Some 60 to 90 species of tRNA exist in bacteria. Each tRNA has a three-nucleotide anticodon as well as a binding site for a cognate amino acid . All tRNAs with a specific anticodon will carry the same amino acid.
• Initiation of translation occurs when the small ribosomal subunit binds with initiation factors and an initiator tRNA at the start codon of an mRNA, followed by the binding to the initiation complex of the large ribosomal subunit.
• In prokaryotic cells, the start codon codes for N-formyl-methionine carried by a special initiator tRNA. In eukaryotic cells, the start codon codes for methionine carried by a special initiator tRNA. In addition, whereas ribosomal binding of the mRNA in prokaryotes is facilitated by the Shine-Dalgarno sequence within the mRNA, eukaryotic ribosomes bind to the 5’ cap of the mRNA.
• During the elongation stage of translation, a charged tRNA binds to mRNA in the A site of the ribosome; a peptide bond is catalyzed between the two adjacent amino acids, breaking the bond between the first amino acid and its tRNA; the ribosome moves one codon along the mRNA; and the first tRNA is moved from the P site of the ribosome to the E site and leaves the ribosomal complex.
• Termination of translation occurs when the ribosome encounters a stop codon , which does not code for a tRNA. Release factors cause the polypeptide to be released, and the ribosomal complex dissociates.
• In prokaryotes, transcription and translation may be coupled, with translation of an mRNA molecule beginning as soon as transcription allows enough mRNA exposure for the binding of a ribosome, prior to transcription termination. Transcription and translation are not coupled in eukaryotes because transcription occurs in the nucleus, whereas translation occurs in the cytoplasm or in association with the rough endoplasmic reticulum.
• Polypeptides often require one or more post-translational modifications to become biologically active.

Contributors and Attributions

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at  https://openstax.org/books/microbiology/pages/1-introduction )

Protein synthesis

Protein synthesis n., plural: protein syntheses Definition: the creation of protein.

Table of Contents

Protein synthesis is the process of creating protein molecules. In biological systems, it involves amino acid synthesis, transcription, translation, and post-translational events. In amino acid synthesis , there is a set of biochemical processes that produce amino acids from carbon sources like glucose .

Not all amino acids are produced by the body; other amino acids are obtained from the diet . Within the cells, proteins are generated involving transcription and translation processes. In brief, transcription is the process by which the mRNA template is transcribed from DNA.

The template is used for the succeeding step, translation. In translation, the amino acids are linked together in a particular order based on the genetic code. After translation, the newly formed protein undergoes further processing, such as proteolysis, post-translational modification, and protein folding.

Proteins are made up of amino acids that are arrainged in orderly fashion. Discover how the cell organizes protein synthesis with the help of the RNAs. You’re more than welcome to join us in our Forum discussion: What does mRNA do in protein synthesis?

Protein Synthesis Definition

Protein synthesis is the creation of proteins. In biological systems, it is carried out inside the cell. In prokaryotes , it occurs in the cytoplasm . In eukaryotes , it initially occurs in the nucleus to create a transcript ( mRNA ) of the coding region of the DNA . The transcript leaves the nucleus and reaches the ribosomes for translation into a protein molecule with a specific sequence of amino acids .

Protein synthesis is the creation of proteins by cells that uses DNA , RNA , and various enzymes . It generally includes transcription , translation , and post-translational events, such as protein folding, modifications, and proteolysis.

The term protein came from Late Greek prōteios , prōtos , meaning “first”. The word synthesis came from Greek sunthesis , from suntithenai , meaning “to put together”. Variant : protein biosynthesis.

Prokaryotic vs. Eukaryotic Protein Synthesis

Proteins are a major type of biomolecule that all living things require to thrive. Both prokaryotes and eukaryotes produce various proteins for multifarious processes and functions. Some proteins are used for structural purposes while others act as catalysts for biochemical reactions.

Prokaryotic and eukaryotic protein syntheses have distinct differences. For instance, protein synthesis in prokaryotes occurs in the cytoplasm. In eukaryotes, the first step (transcription) occurs in the nucleus. When the transcript (mRNA) is formed, it proceeds to the cytoplasm where ribosomes are located.

Here, the mRNA is translated into an amino acid chain. In the table below, differences between prokaryotic and eukaryotic protein syntheses are shown.

Genetic Code

In biology, a codon refers to the trinucleotides that specify for a particular amino acid. For example, Guanine-Cytosine-Cytosine (GCC) codes for the amino acid alanine .

The Guanine-Uracil-Uracil (GUU) codes for valine. Uracil-Adenine-Adenine (UAA) is a stop codon. The codon of the mRNA complements the trinucleotide (called anticodon) in the tRNA.

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mRNA, tRNA, and rRNA

mRNA , tRNA , and rRNA are the three major types of RNA involved in protein synthesis. The mRNA (or messenger RNA) carries the code for making a protein. In eukaryotes, it is formed inside the nucleus and consists of a 5′ cap, 5’UTR region, coding region, 3’UTR region, and poly(A) tail. The copy of a DNA segment for gene expression is located in its coding region. It begins with a start codon at 5’end and a stop codon at the 3′ end.

tRNA (or transfer RNA), as the name implies, transfers the specific amino acid to the ribosome to be added to the growing chain of amino acid. It consists of two major sites: (1) anticodon arm and (2) acceptor stem . The anticodon arm contains the anticodon that complementary base pairs with the codon of the mRNA. The acceptor stem is the site where a specific amino acid is attached (in this case, the tRNA with amino acid is called aminoacyl-tRNA ). A peptidyl-tRNA is the tRNA that holds the growing polypeptide chain.

Unlike the first two, rRNA (or ribosomal RNA) does not carry genetic information. Rather, it serves as one of the components of the ribosome. The ribosome is a cytoplasmic structure in cells of prokaryotes and eukaryotes that are known for serving as a site of protein synthesis. The ribosomes can be used to determine a prokaryote from a eukaryote.

Prokaryotes have 70S ribosomes whereas eukaryotes have 80S ribosomes. Both types, though, are each made up of two subunits of differing sizes. The larger subunit serves as the ribozyme that catalyzes the peptide bond formation between amino acids. rRNA has three binding sites: A, P, and E sites. The A (aminoacyl) site is where aminoacyl-tRNA docks. The P (peptidyl) site is where peptidyl-tRNA binds. The E (exit) site is where the tRNA leaves the ribosome.

Protein Biosynthesis Steps

Major steps of protein biosynthesis:

• Transcription
• Translation
• Post-translation

Transcription is the process by which an mRNA template , encoding the sequence of the protein in the form of a trinucleotide code, is transcribed from DNA to provide a template for translation through the help of the enzyme, RNA polymerase.

Thus, transcription is regarded as the first step of gene expression. Similar to DNA replication, the transcription proceeds in the 5′ → 3′ direction. But unlike DNA replication, transcription needs no primer to initiate the process and, instead of thymine, uracil pairs with adenine.

The steps of transcription are as follows: (1) Initiation, (2) Promoter escape, (3) Elongation, and (4) Termination.

Step 1: Initiation

The first step, initiation, is when the RNA polymerase, with the assistance of certain transcription factors, binds to the promoter of DNA. This leads to the opening (unwinding) of DNA at the promoter region, forming a transcription bubble . A transcription start site in the transcription bubble binds to the RNA polymerase, particularly to the latter’s initiating NTP and an extending NTP . A phase of abortive cycles of synthesis occurs resulting in the release of short mRNA transcripts (about 2 to 15 nucleotides).

Step 2: Promoter escape

The next step is for the RNA polymerase to escape the promoter so that it can enter into the elongation step.

Step 3: Elongation

During elongation, RNA polymerase traverses the template strand of the DNA and base pairs with the nucleotides on the template (noncoding) strand. This results in an mRNA transcript containing a copy of the coding strand of DNA, except for thymines that are replaced by uracils. The sugar-phosphate backbone forms through RNA polymerase.

Step 4: Termination

The last step is termination. During this phase, the hydrogen bonds of the RNA-DNA helix break. In eukaryotes, the mRNA transcript goes through further processing. It goes through polyadenylation , capping , and splicing .

Translation is the process in which amino acids are linked together in a specific order according to the rules specified by the genetic code. It occurs in the cytoplasm where the ribosomes are located. It consists of four phases:

• Activation (the amino acid is covalently bonded to the tRNA ),
• Initiation (the small subunit of the ribosome binds to 5′ end of mRNA with the help of initiation factors)
• Elongation (the next aminoacyl-tRNA in line binds to the ribosome along with GTP and an elongation factor)
• Termination (the A site of the ribosome faces a stop codon)

Post-translation Events

Following protein synthesis are events such as proteolysis and protein folding . Proteolysis refers to the cleavage of proteins by proteases. Through it, N-terminal, C-terminal, or the internal amino-acid residues are removed from the polypeptide.

Post-translational modification refers to the enzymatic processing of a polypeptide chain following translation and peptide bond formation. The ends and the side chains of the polypeptide may be modified in order to ensure proper cellular localization and function. Protein folding is the folding of the polypeptide chains to assume secondary and tertiary structures.

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Further Reading

• Protein Synthesis. (2019). Retrieved from Elmhurst.edu website: http://chemistry.elmhurst.edu/vchembook/584proteinsyn.html
• Protein Synthesis. (2019). Retrieved from Estrellamountain.edu website: https://www2.estrellamountain.edu/faculty/farabee/biobk/BioBookPROTSYn.html
• Protein Synthesis. (2019). Retrieved from Nau.edu website: http://www2.nau.edu/lrm22/lessons/protein-synthesis/protein-synthesis.htm

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The structure of deoxyribonucleic acid

Dna replication, the genetic code and the concept of a gene, transcription, translation of rna into proteins, competing interests, abbreviations, recommended reading and key publications, nobel lectures, review articles, historical perspectives, original research papers, citations for figures, understanding biochemistry: structure and function of nucleic acids.

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Steve Minchin , Julia Lodge; Understanding biochemistry: structure and function of nucleic acids. Essays Biochem 16 October 2019; 63 (4): 433–456. doi: https://doi.org/10.1042/EBC20180038

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Nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), carry genetic information which is read in cells to make the RNA and proteins by which living things function. The well-known structure of the DNA double helix allows this information to be copied and passed on to the next generation. In this article we summarise the structure and function of nucleic acids. The article includes a historical perspective and summarises some of the early work which led to our understanding of this important molecule and how it functions; many of these pioneering scientists were awarded Nobel Prizes for their work. We explain the structure of the DNA molecule, how it is packaged into chromosomes and how it is replicated prior to cell division. We look at how the concept of the gene has developed since the term was first coined and how DNA is copied into RNA (transcription) and translated into protein (translation).

Deoxyribonucleic acid (DNA) is one of the most important molecules in living cells. It encodes the instruction manual for life. Genome is the complete set of DNA molecules within the organism, so in humans this would be the DNA present in the 23 pairs of chromosomes in the nucleus plus the relatively small mitochondrial genome. Humans have a diploid genome, inheriting one set of chromosomes from each parent. A complete and functioning diploid genome is required for normal development and to maintain life.

Discovery and chemical characterisation of DNA

DNA was discovered in 1869 by a Swiss biochemist, Friedrich Miescher. He wanted to determine the chemical composition of leucocytes (white blood cells), his source of leucocytes was pus from fresh surgical bandages. Although initially interested in all the components of the cell, Miescher quickly focussed on the nucleus because he observed that when treated with acid, a precipitate was formed which he called ‘nuclein’. Almost all molecular bioscience graduates would have repeated a form of this experiment in laboratory classes where DNA is isolated from cells. Miescher, Richard Altmann and Albrecht Kossel further characterised ‘nuclein’ and the name was changed to nucleic acid by Altmann. Kossel went on to show that nucleic acid contained purine and pyrimidine bases, a sugar and phosphate. Work in the 1930s from many scientists further characterised nucleic acids including the identification of the four bases and the presence of deoxyribose, hence the name deoxyribonucleic acid (DNA). Erwin Chargaff had found that DNA molecules from a particular species always contained the same amount of the bases cytosine (C) and guanine (G) and the same amount of adenosine (A) and thymine (T). So, for example, the human genome contains 20% C, 20% G, 30% A and 30% T.

DNA is a polymer made of monomeric units called nucleotides ( Figure 1 A), a nucleotide comprises a 5-carbon sugar, deoxyribose, a nitrogenous base and one or more phosphate groups. The building blocks for DNA synthesis contain three phosphate groups, two are lost during this process, so the DNA strand contains one phosphate group per nucleotide.

The structure of DNA

( A ) A nucleotide (guanosine triphosphate). The nitrogenous base (guanine in this example) is linked to the 1′ carbon of the deoxyribose and the phosphate groups are linked to the 5′ carbon. A nucleoside is a base linked to a sugar. A nucleotide is a nucleoside with one or more phosphate groups. ( B ) A DNA strand containing four nucleotides with the nitrogenous bases thymine (T), cytosine (C), adenine (A) and guanine (G) respectively. The 3′ carbon of one nucleotide is linked to the 5′ carbon of the next via a phosphodiester bond. The 5′ end is at the top and the 3′ end at the bottom.

There are four different bases in DNA, the double-ring purine bases: adenine and guanine; and the single-ring pyrimidine bases: cytosine and thymine ( Figure 1 B). The carbon within the deoxyribose ring are numbered 1′ to 5′. Within each monomer the phosphate is linked to the 5′ carbon of deoxyribose and the nitrogenous base is linked to the 1′ carbon, this is called an N-glyosidic bond. The phosphate group is acidic, hence the name nucleic acid.

In the DNA chain ( Figure 1 B), the phosphate residue forms a link between the 3′-hydroxyl of one deoxyribose and the 5′-hydroxyl of the next. This linkage is called a phosphodiester bond. DNA strands have a ‘sense of direction’. The deoxyribose at the top of the diagram in Figure 1 B is not linked to another deoxyribose; it terminates with a 5′ phosphate group. At the other end the chain terminates with a 3′ hydroxyl.

DNA is the genetic material

Although many scientists, including Miescher, had observed that prior to cell division the amount of nucleic acid increased, it was not believed to be the genetic material until the work of Fredrick Griffith, Oswald Avery, Colin MacLeod and Maclyn McCarty. In 1928, Griffith showed that living cells could be transformed by extracts from heat-killed cells and that this transformation had the potential to permanently change the genetic makeup of the recipient cell. Griffith was working with two strains of the bacterium Streptococcus pneumoniae. The encapsulated so-called S strain is virulent, whereas the non-capsulated R strain is nonvirulent. If the S strain is injected subcutaneously into mice, the mice die, whereas, if either live R strain is injected or heat-killed S strain is injected, the mouse lives. However, if a mixture of live R strain and heat-killed S strain is injected into a mouse, the mouse will die, and live S strain can be isolated from the blood. So, in the Griffith experiment a component of the heat-killed S strain is transforming the R strain. In 1944, Avery, MacLeod and McCarty went on to show that it was DNA that could transform the avirulent bacterium. They isolated a crude DNA extract from the S strain and destroyed any protein, lipid, carbohydrate and ribonucleic acid (RNA) component and showed that this purified DNA could still transform the R strain. However, when the purified DNA was treated with DNAse, an enzyme that degrades DNA, transformation was lost.

Alfred Hershey and Martha Chase confirmed that DNA was the genetic material. They used a virus that infects bacteria called a bacteriophage. The bacteriophage contains a protein capsid surrounding a DNA molecule. They showed that when bacteriophage T2 infects Escherichia coli , it is the phage DNA, not protein, that enters the bacterial cell.

Determining the structure of DNA

Once it had been shown that DNA was the genetic material, there was a race to determine the three-dimensional structure of the DNA molecule. At King’s College London, Rosalind Franklin and Maurice Wilkins, having obtained data using X-ray diffraction, had proposed that DNA had a helical structure and Franklin had obtained a particularly good X-ray diffraction pattern. In Cambridge, James Watson and Francis Crick used model building together with data from a variety of sources including Franklin’s X-ray diffraction pattern and Chargaff’s base composition data to work out the now well-known double helix structure of DNA. Their work was published in Nature in 1953. The Watson–Crick structure is shown in Figure 2 A.

DNA structure

( A ) The DNA double helix, with the sugar phosphate backbone on the outside and the nitrogenous bases in the middle. ( B ) An A:T and a G:C base pair with the C1′ of the deoxyribose indicated by the arrow. Note that the C1′ of the deoxyribose is in the same position in all base pairs. In this figure, the atoms on the upper edge of the base pair face into the major groove and those facing lower edge face into the minor groove. The hydrogen bonds between the base pairs are indicated by the dotted line.

DNA is a two-stranded helical structure, the two strands run in opposite directions. In Figure 2 A, one strand is running 5′ to 3′ top to bottom, whereas the other strand is running 3′ to 5′ top to bottom. The helix is right-handed which means that if you are looking down the axis, the helix turns clockwise as it gets further away from you. The two chains interact via hydrogen bonds between pairs of bases with adenine always pairing with thymine, and guanine always pairing with cytosine. The Watson–Crick structure therefore accounts for and explains the Chargaff data which showed that there was always an equal amount of C and G and of A and T. The regular nature of the double helix comes about because the distance between the 1′ carbon of the deoxyribose on one strand and 1′ carbon of the opposite deoxyribose is always the same irrespective of the base pair ( Figure 2 B). The 1′ carbons of the deoxyribose opposing nucleotides do not lie directly opposite each other on the helical axis, this means that the two sugar–phosphate backbones are not equally spaced along the helical axis resulting in major and minor grooves.

The diameter of the helix is 2 nm, adjacent bases are separated by 0.34 nm (0.34 × 10 −9 m) and related by a rotation of 36°, this results in the helical structure repeating every 10 residues. DNA molecules are normally very long and the sequence of bases along the DNA chain is not restricted. For example, the genome of the bacterium E. coli is a single circular chromosome which contains 4.6 million base pairs (4.6 × 10 6 bp), this is therefore 1.6 mm long (4.6 × 10 6 × 0.34 × 10 −9 m). The human genome is made up of 24 distinct chromosomes, chromosomes 1–22 and the X and Y chromosomes present in the nucleus plus mitochondrial DNA. The nuclear chromosomes vary in size from approximately 50–250 × 10 6 bp, the mitochondrial DNA is 17 × 10 3 bp. The total length of a haploid human genome is 3 × 10 9 bp. Within a single human diploid cell, which contains 23 chromosome pairs there is 2 m of DNA. Based on the assumption that humans contain 3 trillion cells with a nucleus, if all the DNA from a single human individual was put end to end, it would reach to the sun and back approximately 20 times.

Another important class of nucleic acids is RNA, the roles of RNA molecules in the cell will be discussed below. Chemically RNA is similar to DNA, it is a chain of similar monomers. The building blocks are nucleotides containing the 5-carbon sugar ribose, a phosphate and a nitrogenous base. The phosphate is attached to the 5′ carbon of the ribose and the nitrogenous base to the 1′ carbon ( Figure 3 ). RNA contains four bases adenine, guanine, cytosine and uracil. RNA is more labile (easily broken down) than DNA and most RNA molecules do not form stable secondary structures, some notable exceptions will be discussed below. The properties of RNA make it ideal as a genetic messenger during protein synthesis, the idea of this genetic messenger, mRNA, was proposed by François Jacob and Jacques Monod.

The structure of RNA

An RNA strand containing the four nucleotides with the nitrogenous bases: adenine (A), cytosine (C), guanine (G) and uracil (U) respectively. The 3′ carbon of the ribose of one nucleotide is linked to the 5′ carbon of the next via a phosphodiester bond. The 5′ end on the left and the 3′ end on the right.

Packaging of DNA into eukaryotic cells

DNA has to be highly condensed to fit into the bacterial cell or eukaryotic nucleus. In eukaryotes, histone proteins are used to condense the DNA into chromatin. The basic structure of chromatin is the nucleosome, a nucleosome contains DNA wrapped almost two times around the histone octamer (comprising two copies each of the histone proteins H2A, H2B, H3 and H4) ( Figure 4 ). Further levels of compaction are required to fit the DNA into the nucleus ( Figure 4 ), the nucleosomes are folded upon themselves to form the 30-nm fibre, this is then folded again to form the 300-nm fibre and during mitosis further compaction can occur forming the chromatid which is 700 nm in diameter.

The different levels of chromatin structure

Histone proteins (H2A, H2B, H3 and H4) associate to form a histone octamer. Approximately 147 bp of DNA wraps around histone octamer to form a nucleosome, generating a ‘beads on a string’ structure, the nucleosome together with histone H1 condense into the 30-nm fibre, there is further condensation to form the 300-nm fibre. During mitosis there is further compaction (not shown).

Processes such as DNA replication and DNA transcription need to occur in the chromatin environment and because of the level of compaction, this acts as a barrier to proteins that need to interact with DNA. Therefore, chromatin structure plays an important role in processes such as regulation of gene expression in eukaryotes. DNA and the histone proteins can be chemically modified, these are called epigenetic modifications as they do not change the DNA sequence, however, they can be passed on during cell division and to subsequent generations, a process known as epigenetic inheritance. As these epigenetic modifications can alter the chromatin structure they regulate gene transcription and can affect the phenotype. Epigenetics plays key roles in many processes, including development, cancer and behaviour and addiction. This will be discussed further later in this article.

Nuclear organisation plays an important role in many biological processes including regulation of gene transcription. In recent years the development of several techniques, including microscopy, have allowed us to gain an understanding of the way the genome is organised in 3D. Individual chromosomes are not randomly spaced within the nucleus; each chromosome has a distinct territory. Actively transcribed regions from different chromosomes are often close to each other and near the interior of the nucleus, whereas, inactive genes are on the periphery or near a special area called the nucleolus where ribosomal RNA is transcribed.

Whenever a cell divides there is a need to synthesise two copies of each chromosome present within the cell. For example in a human, prior to cell division, all 23 pairs of chromosomes need to be replicated to form 46 pairs, so that following cell division each daughter cell has a full complement (23 pairs) of chromosomes. The structure of DNA gives us a clue to how it is replicated, this was eloquently postulated by Watson and Crick in their 1953 paper: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”. Each strand can act as a template for the synthesis of the complementary strand, so the replication machinery would ‘unzip’ the double helix and read along the two existing ‘parent’ strands, synthesising a complementary new ‘daughter’ strand with A opposite T, C opposite G etc. This is described as semi-conservative, since each ‘new’ double-stranded DNA molecule has one original parent strand and one newly made daughter ‘strand’.

The evidence that DNA replication was semi-conservative came from an elegant experiment completed by Matthew Meselson and Franklin Stahl. They labelled the parental DNA with a heavy isotope of nitrogen ( 15 N) by growing bacteria in a growth medium that contained 15 NH 4 Cl. They then grew the bacteria, in a medium that contained 14 NH 4 Cl, in conditions such that any newly synthesised DNA would contain 14 N. Since DNA replication is semi-conservative, after one round of DNA replication, each cell would have a DNA molecule that contains one ‘old’ parental strand labelled with 15 N and one ‘new’ daughter strand labelled with 14 N. This was shown by analysing the density of the DNA using density-gradient centrifugation. As predicted, they observed that the new daughter DNA molecule had a density consistent with the fact that it contained both 15 N and 14 N and that this daughter DNA contained one strand with 15 N and another strand with 14 N.

DNA polymerase and DNA synthesis

The enzyme, DNA polymerase, is responsible for DNA synthesis. DNA polymerase is a template-driven enzyme, so it will use the parental DNA strand as a template. It cannot synthesise DNA in the absence of a template. In addition, it will only add nucleotides on to the 3′ end of an existing nucleic acid chain. The building blocks for DNA synthesis are deoxynucleoside triphosphates (dATP, dTTP, dCTP and dGTP). During DNA synthesis, the base within the incoming deoxynucleoside triphosphate pairs with the complementary base on the template strand, a phosphodiester bond is formed between the 5′ phosphate on the incoming nucleotide and the free 3′ hydroxyl on the existing nucleic acid chain; pyrophosphate is released ( Figure 5 ).

DNA synthesis

( A ) DNA polymerase binds the template DNA and the new strand. The next nucleotide to be added to the 3′ end of the growing chain will contain guanine (G), this is complementary to the C on the template strand. DNA polymerase catalyses the formation of a phosphodiester bond. ( B ) The chemical reaction during the formation of a phosphodiester bond, showing the addition of a nucleotide containing guanine and the release of pyrophosphate.

Pyrophosphate is the two phosphate residues within the deoxynucleoside triphosphate building block that are not incorporated into the DNA chain. DNA polymerase synthesises DNA in the 5′ to 3′ direction, because it can only add nucleotides on to the 3′ end of the chain. DNA polymerase has proofreading activity, so after the phosphodiester bond has been formed, the base pairing is checked and if a nucleotide with an incorrect base has been added, DNA polymerase will remove the nucleotide using a 3′ to 5′ exonuclease activity. Exonucleases are enzymes that can remove nucleotides from the ends of a DNA molecule, 3′ to 5′ exonucleases remove nucleotides from the 3′ end of a DNA molecule and therefore can remove the last nucleotide that was added during DNA replication. This is analogous to using the delete key to remove a letter that you have typed incorrectly before adding the correct one and continuing typing.

DNA polymerase requires a short double-stranded region with a free 3′ hydroxyl in order to start making a copy of the template; this ensures that DNA is synthesised in a controlled way. Initiation of DNA synthesis uses a small RNA primer (8–12 bases) made by the enzyme primase. DNA polymerase will then extend from the primer copying the template and synthesising the daughter DNA strand. This means that when DNA synthesis first starts each DNA molecule actually contains a small piece of RNA at its 5′ end. This RNA will ultimately be replaced with DNA, how this is done is discussed below.

The origin of replication and the replisome

A large multiprotein complex, called the replisome, is responsible for DNA replication. In prokaryotes, two replisomes form at a specific point on the chromosome called the Origin of Replication ( ori ). The DNA in this region will be opened up, ‘unzipped’ so that the replication machinery can gain access to single-stranded parental DNA, which will act as template for synthesis of the new daughter strands. The two replisomes then travel in opposite directions around the circular prokaryotic chromosome, each replisome forming a replication fork, a schematic representation of one replication fork is shown in Figure 6 .

DNA synthesis at a replication fork

A single replication fork showing the leading and lagging strands. The leading strand is synthesised continuously, reading the template 3′ to 5′, synthesising DNA in the 5′ to 3′ direction. The lagging strand is synthesised discontinuously, in short Okazaki fragments (1000 bases in prokaryotes and 100 bases in eukaryotes).

The replication fork

Within the replication fork, on the so-called leading strand, DNA polymerase moves 3′ to 5′ with respect to the template and synthesises DNA in the 5′ to 3′ direction as it moves in the same direction as the replication fork. Although overall the lagging strand is synthesised in the 3′ to 5′ direction, it is actually synthesised discontinuously in small segments called Okazaki fragments, which are synthesised 5′ to 3′ ( Figure 6 ). Each Okazaki fragment will be started with an RNA primer and is synthesised in the opposite direction to the movement of the replication fork. In prokaryotes, Okazaki fragments are 1000–2000 bases in length. In Figure 6 you will see that the DNA polymerase synthesising the Okazaki fragment will eventually reach the primer for the previous Okazaki fragment. When this happens the primer for the previous fragment is removed by a DNA polymerase using 5′ to 3′ exonuclease activity. DNA polymerase then replaces the missing nucleotides by adding them to the 3′ end of the last Okazaki fragment. When all the primer has been removed, there will be two DNA strands adjacent to each other but not joined by a phosphodiester bond, these two strands are joined together by the enzyme DNA ligase.

The replisome contains a number of other important proteins required for DNA replication. The double-stranded DNA needs to be separated, ‘unzipped’, by a helicase to generate the single-stranded DNA templates for DNA polymerase. As the replication fork moves along the helical DNA, the coils in the DNA in front of the fork become compressed so the DNA is described as being overwound; a topoisomerase is required to ‘relax’ it by remove the over-winding. Single-stranded binding proteins (SSBs) bind the lagging strand template to stabilise and protect the single-stranded DNA.

The two replication forks that form at the ori will move in opposite directions around the circular prokaryotic genome until they reach the terminator sequence, ter , which is on the opposite side of the genome compared with the ori , i.e. it is at 6 o’clock compared with 12 o’clock. This results in the complete replication of the genome. Once DNA replication has been completed a post-replication DNA repair process will correct errors that were not corrected by the proofreading activity of DNA polymerase. The fidelity of DNA replication is extremely high, resulting in an error rate of 1 mistake per 10 9 –10 10 nucleotides added.

DNA replication in eukaryotes

DNA replication is essentially the same in eukaryotes and prokaryotes. In both cases two replisomes form at an ori and generate two replication forks moving in opposite directions away from the origin. In each replication fork there are leading and lagging strands. There are two major differences. The first is that, due to the larger genome size, each chromosome has multiple origins of replication, so there will be a large number of replication forks on each chromosome.

The second difference is that, with the exception of mitochondrial DNA, eukaryotic chromosomes are linear and this results in an issue because of lagging strand synthesis. Replication of a linear chromosome results in shortening of one 5′ end of each daughter DNA molecule. This is because when the primer required for the last Okazaki fragment is removed, DNA polymerase cannot fill the gap ( Figure 7 A). Repeated rounds of DNA replication results in shorter and shorter DNA molecules. If this is not corrected, eukaryotes would have become extinct as their chromosomes get shorter with each generation. Eukaryotes have a mechanism to preserve the ends of chromosomes when it counts; that is in the gametes. The terminal ends of chromosomes, telomeres, contain a highly repeated sequence, for example, in humans the sequence TTAGGG is repeated in tandem 100 to over 1000-times. Repeated rounds of DNA replication will result in the shortening of these telomeric sequences that is the number of repeats will reduce. Telomerase, an RNA containing enzyme, can add additional copies of the repeat sequence to the 3′ end, replacing those lost during DNA replication (see Figure 7 ).

Telomeres and telomerase

( A ) Following DNA replication and removal of the primer for the last Okazaki fragment of the lagging strand, there will be a region at the 3′ end that is not base paired, called a 3′ overhang. ( B ) Telomerase binds and uses the RNA it contains to act as a template to extend the 3′ overhang. This extends the 3′ end sufficiently for a new RNA primer to bind and the final Okazaki fragment to be made.

This actually extends the 3′ end of the telomere rather than extending the 5′ that is initially lost during DNA replication. The RNA sequence within telomerase is complementary to the 3′ telomeric sequence and so can bind and act as a template for synthesis of a short DNA sequence. Telomerase then moves along the newly synthesised strand and the process is repeated. Multiple rounds of elongation and translocation ultimately results in the 3′ end being extended so that it is long enough for it to act as template for synthesis of another Okazaki fragment, hence extending both strands of the telomere. Only germ cells and a few other actively dividing cells (e.g. haematopoietic cells) have sufficient levels of telomerase activity to counteract the loss of repeat sequences during DNA replication. At birth, telomeres are over 10000 base pairs in length and there are enough repeats to allow DNA replication and somatic cell division during the lifetime of the organism. If telomeres become too short this will trigger programmed cell death (a process called apoptosis). The lack of telomerase activity in somatic cells limits the number of cell divisions that can occur, and this is a ‘problem’ that needs to be overcome by cancer cells. Telomerase activity is reactivated in most cancers, allowing these cells to divide indefinitely and therefore this activity is a potential target for cancer therapies.

An understanding of DNA synthesis is central to many experimental approaches in molecular biosciences, it allows us to determine DNA sequences including that of the human genome, to analyse environmental samples to better understand the living world around us and to analyse minute biological samples from crime scenes to identify offenders. It is exploited in medicine, for example several drugs used to treat HIV infection or exposure are nucleoside analogues that inhibit DNA synthesis. Many chemotherapy agents used to treat cancer target DNA replication.

As we have seen in the previous two sections, the genetic material in a cell is made of DNA and can be copied and passed on to progeny through DNA replication allowing for inheritance of the information that it carries. A large proportion of the information on the DNA is first transcribed into mRNA and then translated into proteins. However there are some RNAs that are never translated into proteins and these have important functions too. Phrases like ‘it is in my genes’ or ‘in my DNA’ are used in common speech to mean to be an important part of who someone is.

The term gene was coined in the early 1900s to describe the basic unit of heredity. Genes were thought of as distinct loci arranged lineally on chromosomes. Breeding experiments with the fruitfly Drosophila supported this view and showed that if two genes are close together on a chromosome they are more likely to be inherited together. The observation that mutations in genes could give rise to altered phenotypes gave rise to the ‘one gene one polypeptide’ hypothesis. Once it became clear that genes were made of DNA, what is referred to as the central dogma of molecular biology was coined. This describes a two step process in which the genes on the DNA are transcribed into RNA and then translated into a sequence of amino acids that makes up a protein. The information flow is from DNA to RNA and then to protein ( Figure 8 ).

The flow of genetic information

The arrows represent steps where DNA or RNA is being used as a template to direct the synthesis of another polymer, either RNA or protein.

However there are exceptions to this, firstly some viruses have RNA genomes and in some cases these are reverse transcribed into DNA before the genes can be expressed. The retrovirus HIV is an example of this. The other exception is that not all functional RNAs are translated into proteins (see non-coding RNAs below).

The genetic code

The genetic code is the set of rules used by living cells to translate the information encoded within genetic material into proteins. When DNA and RNA were first discovered, the relative simplicity of nucleic acids led many scientists to doubt that it carried the genetic information. DNA only has four different kinds of bases; the question was how it could code for 20 amino acids. If there were a 1:1 correlation between bases and amino acids DNA could only encode four amino acids. Pairs of bases would give 16 possible combinations which is still not enough. However if you consider a triplet code you have 64 possibilities, which is more than enough. This is the code that we are familiar with where each codon, a sequence of three nucleotides, specifies a particular amino acid. This triplet code still did not seem logical because now you have far more codons than you need. There are some other important questions about the genetic code too; are the spare codons used? Is the code overlapping? And is it continuous or are there spacers indicating the end of each codon?

Table 1 shows the genetic code as we now understand it. It is written as RNA with a U rather than a T because it is RNA that cells translate into amino acids. The code is said to be redundant or degenerate because a single amino acid is often coded for by more than one codon. In most cases it is the third nucleotide in the codon that differs; this is often referred to as the degenerate position.

Evidence for the triplet code

The experiments that allowed scientists to decipher the genetic code were carried out long before we were able to determine the sequence of DNA. While it was possible at that time to determine the proportions of each different amino acid in a protein, it was not yet possible to work out the order in which they occurred. Francis Crick and Sydney Brenner answered some key questions with an experiment using mutants of a virus that infects bacteria called bacteriophage. The normal or wild-type phage will infect E. coli and grow. Crick and Brenner investigated mutants that would not grow on some strains of E. coli .

Mutants which are insertions or deletions cause what are called frameshifts. Inserting a single adenine base into the DNA sequence not only changes the amino acid at the position of the insertion but all subsequent amino acids translated from that sequence (Compare Figures 9 A and B); the reading frame has been shifted by one base and it results in a protein that is non-functional. However if you insert three nucleotides you often get a wild-type or near wild-type phenotype. This is because you have inserted a whole triplet codon, you will get one or two amino acids that were not in the original sequence but the reading frame is not shifted ( Figure 9 C) and the rest of the sequence is normal.

DNA sequence showing the amino acid translation underneath

( A ) Wild-type sequence, ( B ) a single base insertion (shown in red) causes a frameshift so all subsequent amino acids are different from the wild-type, ( C ) insertion of three base pairs (shown in red) causes two incorrect amino acids to be incorporated into the protein but there is no frameshift so the rest of the protein has the wild-type sequence.

Crick and Brenner were looking for what they called suppressor mutations that would rescue the mutant and allow it to grow normally. They showed that their suppressor mutants did not simply reverse the original mutation; they often added or subtracted one or more bases. They worked out that if you insert or delete one, two or four nucleotides then you see a mutant phenotype. However, if you insert or delete three nucleotides, this has little or no effect. This was strong supporting evidence for a triplet code. This is also evidence for a redundant code where the same amino acid can be coded in more than one way. If the code were non-redundant there would be 20 codons that code for amino acids and 44 that are ‘nonsense’ codons. In this case inserting three nucleotides would be most likely to introduce a nonsense codon and not restore the wild-type. Crick and Brenner proposed correctly that the genetic code is read from a fixed starting point and the bases are read in groups of three.

Cracking the code

At about the same time two American scientists Marshall Nirenberg and Heinrich Matthaei had developed a cell-free system which could synthesise proteins in a test tube when provided with an RNA molecule. They showed that when provided with an artificial RNA chain composed only of uracil (polyuracil) the system made a polypeptide composed entirely of phenyalanine residues. They now had a tool that they could use to crack the genetic code. RNA composed of cytosine (C) residues directed the synthesis of polyproline and RNA composed of adenosine (A) made polylysine. Experiments with combinations of nucleotides demonstrated that, for example, if you make RNA from A and C you produce proteins containing only six amino acids: asparagine, glutamine, histidine, lysine, proline and threonine. There are eight possible triplet codons that can be made from A and C, two of these we know encode proline and cysteine. The remaining four amino acids must be encoded by other combinations of A and C. This of course provides additional evidence for the redundancy of the genetic code.

These experiments using RNA molecules composed of random combinations of two or three bases were not enough to fully crack the genetic code. The use of chemically synthesised RNA molecules of known repeating sequence added some more important information. For example a synthetic RNA of alternating A and G residues (AGAGAGAGAG…) can be read as two alternating codons CAC and ACA. It encodes a protein of alternating histidine and threonine residues.

In the last section, we will discuss how tRNAs and ribosomes decode the genetic code and synthesise proteins. The final detail of the genetic code was determined by a technique using ribosome-bound tRNAs. Pieces of RNA as short as a single codon will bind to ribosomes and if amino acids attached to tRNA are added they will associate with the complementary RNA. If you then filter the solution you trap only the tRNAs that are bound to the ribosome, these are the ones specified by the codon in your RNA.

Start and stop codons

Of the 64 possible codons, 61 encode amino acids. The three remaining codons: UAA, UAG and UGA do not code for an amino acid, they are sometimes called nonsense codons. They are stop codons; when the ribosome encounters these protein synthesis stops. The AUG codon encodes the amino acid methionine but it is also the most common start codon. As you will see in the last section, the first residue in eukaryotic proteins is always a methionine and in prokaryotes it is a modified amino acid N-Formylmethionine.

Expanding the genetic code

Nature uses a small set of amino acids to make proteins, however if we were able to engineer cells that could use a wider range of building blocks with different physical and chemical properties it would be possible to make novel materials some of which could have useful therapeutic properties; this is one of the aims of synthetic biology. To do this successfully we need to reprogramme the genetic code and to engineer the translation machinery (see later section) to use these new combinations. Some progress has been made, for example in using both the UAG stop codon and the AAG codon for arginine to code for amino acids not normally found in proteins.

Current concept of the gene

Once the genetic code was cracked it was clear that a gene is a sequence of bases on a DNA molecule that codes for a sequence of amino acids in a polypeptide chain or for an RNA molecule with a specific function. The availability of DNA sequences (see ‘Recombinant DNA Technology and DNA Sequencing’ in this issue of Essays in Biochemistry ) of individual genes made it possible to look for patterns characteristic of genes. A gene that codes for a protein has a start codon followed by a series of codons that encode the amino acid sequence and then a stop codon; this is called an open reading frame.

Whole genome sequencing has provided biological data on an unprecedented scale. The need to analyse sequence data has led to the development of the field of bioinformatics; the analysis of these data to answer biological questions. One key concept used in bioinformatics is that of homology. Two organisms that have a common ancestor are said to be homologous and the same can be said of a structure or of a gene. For example limbs with five digits (the pentadactyl limb) are found not only in humans and other mammals but also in birds, reptiles and amphibians. The limbs are homologous, and this is evidence of a common evolutionary ancestor of all of these groups of animals. The same is true of genes. All vertebrates have red blood cells that contain haemoglobin, adult human haemoglobin is made from two α and two β globin molecules. The DNA sequence of the genes that encode globin molecules in vertebrates are all similar to each other and you can estimate how long ago two animals shared a common ancestor by looking at how similar their globin genes are. This principle can also be used to find genes in a new piece of DNA sequence; if there is a section of sequence that is similar to a known gene then it is likely to encode a homologous gene.

A gene is more than just the sequence that encodes the protein; it also includes sequences involved in regulation of gene expression such as promoter sequences that define where transcription starts and are the sites where proteins involved in transcription bind to the DNA. In bacteria, almost all genes are a single uninterrupted sequence of DNA. In eukaryotes the situation is more complicated because the coding region is usually interrupted by introns. The primary transcript is referred to as precursor or pre-mRNA, this contains both exons and introns. The introns are removed when the pre-mRNA is processed before it leaves the nucleus ( Figure 10 ) leaving the exons which are spliced together to make the mature mRNA. Eukaryotic mRNAs have a 5′ cap which is a methylated guanosine nucleotide added to the 5′ end of the mRNA by an unusual 5′ to 5′ linkage; this is important in initiating translation. At the 3′ end is the poly A tail, this is a chain of between 100 and 250 adenine residues added to the mRNA to increase its stability. Analysis of the human genome sequence suggests that there are approximately 20000–25000 protein-coding genes, however there are far more different proteins. This is because many genes are capable of encoding several variants of a protein. Alternative splicing allows for different combinations of exons to be included in the mature mRNA and genes can also have several alternative promoters and alternative poly A sites. It is thought that 95% of human genes are alternatively spliced.

The structure of a protein-coding eukaryotic gene

The DNA includes an untranslated region at both the 5′ and 3′ ends as well as introns and exons. The codon where translation starts (green) and the stop codon (red) are shown. The DNA is transcribed into mRNA and is processed by addition of the 5′ cap, splicing out the introns and addition of the poly A tail. This mature mRNA is exported from the nucleus into the cytoplasm.

Non-coding RNAs

Only approximately 1.2% of the human genome codes for protein. However, if you compare the genomes of the human, the mouse and the dog you can see that much more of the genome is under what is called negative selection since the species diverged. Negative selection means that mutations which are disadvantageous are selected against. This suggests that more than just the protein coding regions affect the fitness of the organism carrying the DNA. Some of these are DNA sequences that are important in controlling gene expression (next section). However systematic screens are revealing large numbers of RNA transcripts that are processed but do not encode proteins. The most well-known are transfer RNAs and ribosomal RNAs both of which as we will see in a later section are fundamental to protein synthesis. However we are beginning to understand that there are other non-coding RNAs that carry out important cellular processes.

Two types of non-coding RNA, small inhibitory RNAs (siRNAs) and microRNAs (miRNAs) have a role in reducing gene expression after the mRNA has been transcribed from the DNA. They work by targeting a protein complex called RISC to specific mRNAs which it then degrades. Expression of the gene is specifically knocked out or reduced and the phenotypic effect of this can then be observed. Another group of non-coding RNAs play an important role in increasing the stability and correct folding of ribosomal RNAs. This process takes place in a compartment within the nucleus called the nucleolus; the RNAs are called small nucleolar RNAs (snoRNAs). These are mostly generated from intron RNA after it has been spliced out of the precursor mRNA and they function in association with proteins.

Modern concept of a gene

The modern concept of the gene has to take into account all of the complexity of mRNA processing including alternative splicing, regulatory sequences and polyadenylation sites as well as the plethora of non-coding RNAs. A definition of a gene that takes these factors into account would be that a gene codes for one or more transcripts that can function as an RNA or can be translated into one or more proteins.

We have seen that a gene can encode either an RNA product or a protein sequence. The production of both requires the gene to be transcribed into RNA, either because the RNA is the final product or because the RNA will need to act as template for protein synthesis. RNA synthesis is very similar in prokaryotes and eukaryotes, being catalysed by the enzyme RNA Polymerase. However, of the processes discussed in this article it is arguably the one that differs most between prokaryotes and eukaryotes. One difference is that in eukaryotes the whole process needs to occur in a chromatin context, so access to the DNA template is limited. Regulation of gene expression is a major facilitator of cell differentiation, homoeostasis and speciation. Different cell types turn on transcription of different genes giving rise to their differentiated phenotypes. If we look at mammals as an example of speciation, they all have roughly the same gene content; it is how transcription is regulated that has changed as mammals have evolved. For example, if you compare humans and mice, the important changes to the human and mouse genome sequence that have occurred since they diverged from a common ancestor, are predominantly in the sequences that control transcription rather than in protein coding sequences.

RNA polymerase

DNA-dependent RNA polymerases are responsible for transcription of DNA into RNA. Like DNA Polymerase, RNA polymerase requires a DNA template and nucleoside triphosphate precursors. RNA polymerase does not require a primer. During RNA synthesis, the base within the incoming nucleoside triphosphate pairs with the base on the DNA template, a phosphodiester bond is formed, and pyrophosphate is released. RNA polymerase synthesises RNA in the 5′ to 3′ direction, because it can only add nucleotides on to the 3′ end of the chain. During transcription only one DNA strand is transcribed into RNA.

Gene transcription

When a gene is transcribed, RNA polymerase will bind upstream from the start of the gene, it will unwind almost two turns of the DNA helix to form a transcription bubble, it will add nucleotides on to the growing RNA chain, the last 12 nucleotides to be added to the RNA chain will base pair with the DNA template, forming a DNA–RNA heteroduplex ( Figure 11 ).

Schematic diagram of transcription

As each nucleotide is added to the growing chain, the transcription bubble and the heteroduplex moves with respect to the DNA template. So, as RNA polymerase synthesises RNA, there is unwinding of the DNA template in front of the site of synthesis and rewinding of DNA once RNA polymerase has passed through. Once RNA polymerase has transcribed the gene, transcription will terminate. For some genes, transcription termination is signalled by a particular sequence within the DNA, a terminator sequence, which RNA polymerase recognises. In some cases, RNA polymerase requires the help of other protein factors to recognise the terminator sequence. Finally, many eukaryotic genes do not contain a specific terminator sequence; instead, termination of transcription is linked to other events, for example cleavage of the RNA prior to addition of the polyA tail. Termination of transcription, leads to the dissociation of RNA polymerase from the DNA template and release of the RNA product. In prokaryotes, mRNA does not need processing before it can be translated, in fact, as will be discussed below, mRNA is translated as it is being made. However, the initial transcript in eukaryotes does need to be processed to produce a functional mRNA that can be exported to the cytoplasm for translation.

Control of transcription in prokaryotes

At many genes in prokaryotes, RNA polymerase can bind to the gene and initiate transcription without other protein factors. However, for most prokaryotic genes, the binding of RNA polymerase to the gene is controlled by transcription factors to ensure the correct genes are transcribed at the correct level within the cell. Upstream from the transcription start there will be a ‘promoter’ which contains specific DNA sequences that are recognised by RNA polymerase and transcription factors. Each gene will have a different promoter sequence and can be controlled by different transcription factors. A good example of this type of promoter is the promoter that controls the lac operon in E. coli ( Figure 12 ). Transcription factors that up-regulate transcription are called activators and those that down-regulate transcription are called repressors. In this example RNA polymerase on its own can bind the promoter and drive low levels of transcription. If the repressor binds it will stop all transcription and would override RNA polymerase and the activator. In the absence of the repressor, if the activator is present then it can drive high levels of transcription.

The different binding sites for transcription factors are shown on the DNA; ABS, activator binding site, RBS, repressor binding site. The left-hand panel indicates the presence of lactose and/or glucose in the environment, the right-hand panel indicates transcription levels.

The lac operon codes for genes required to use lactose and needs to be controlled in response to glucose and lactose concentrations. A repressor protein is responsible for responding to lactose concentration and an activator is responsible for responding to glucose ( Figure 12 ).

In the absence of lactose, the lac operon is kept in an off state by the repressor protein binding to the promoter and stopping transcription. If lactose is present in the cell, it will bind to the repressor and this stops the repressor binding the promoter, RNA polymerase can bind and drive low levels of transcription. If the cell is starved of glucose, the activator is turned on and this binds the promoter and helps RNA polymerase to initiate transcription, resulting in high rates of transcription.

In the examples above, RNA polymerase on its own drives low levels of transcription. This might not be the case for all promoters, at some promoters RNA polymerase may not be able to bind and drive transcription without an activator protein. At other promoters RNA polymerase on its own will be able to drive high levels of transcription and a repressor protein would be needed to turn off transcription.

Control of transcription in eukaryotes

Control of transcription in eukaryotes has to occur on a chromosome which is condensed into chromatin ( Figure 13 ). In addition, transcription requires the assembly of a large multiprotein complex at the gene. This complex will contain RNA polymerase and several other general transcription factors (GTFs). The core promoter is a region that overlaps the transcription start and is the binding site for RNA Polymerase and the GTFs. In addition, there will be further control sequences, enhancers, that can be just upstream or several 1000 base pairs away from the core promoter. In the absence of activator proteins the chromatin structure will stop RNA polymerase and the GTFs binding to the core promoter. Here histone proteins act as generic repressors of transcription. In order for a transcription to be turned on activators will bind the enhancers and recruit co-activators which open up the chromatin structure and ensure the core promoter is not blocked by histone proteins. The activators and co-activators will then assemble RNA polymerase and the GTF at the core promoter and drive transcription initiation. Transcription factors will also ensure the chromatin structure across the whole gene is in a conformation that is suitable for transcription.

Regulation of transcription in eukaryotes

( A ) When a gene is in a silent state the surrounding DNA will be in condensed chromatin and the histones will epigenetic modifications which facilitate gene repression (red spheres). ( B ) A gene that is being transcribed will have activators bound to enhancer sequences, the activators recruit co-activators that acetylate the histone and add other epigenetic modifications that facilitate gene transcription (green spheres). The activator and co-activators will recruit RNA Polymerase and the GTFs to the core promoter.

Repressors are not normally required to block assembly of transcription complex at the core promoter, however, they are important in the regulatory patterns needed in complex multicellular organisms. Eukaryotes have repressor proteins which can block the action of a specific activator and ensure the activator is only active when required. Repressors can work in a number of ways including binding to DNA and blocking the binding of the activator to the DNA, stopping the activator interacting with other proteins required for transcription or by binding to the activator and keeping the activator in the cytoplasm.

Epigenetics

As discussed above transcription initiation in eukaryotes requires the opening up of the chromatin structure. This is facilitated by co-activator proteins that can move the relative position of the nucleosomes ( Figure 4 ) with respect to the DNA and hence make certain regions of the DNA more accessible. They can also add chemical tags to both the histone proteins and DNA ( Figure 13 ). These epigenetic modifications can affect whether a gene or genomic region is available for transcription or is transcriptionally silenced. Histones are acylated by enzymes which transfer an acetyl functional group to from acetyl-coenzyme A to lysine residues in the histone protein. This is linked to activation of transcription because it reduces the positive charge on histones and therefore reduces their affinity for the negatively charged DNA. Acetylation can also act as a tag that is recognised by other proteins that drive gene transcription. This modification of the DNA is described as epigenetic because it affects gene expression rather than the genetic code itself. Conversely some repressor proteins will recruit co-repressors that deacetylate histones, increasing their affinity for DNA causing the chromatin to be highly condensed and leading to transcriptional silencing. Methylation of lysine residues is another epigenetic tag, a single lysine residue can have 1, 2 or 3 methyl groups added. Unlike acetylation, methylation of lysine residues does not change the positive charge. The consequences of histone methylation are more complex because depending on which lysine residue is methylated and the level of methylation, the tag may mark that region of the genome for transcription activation or repression.

DNA methylation is another important epigenetic modification which leads to transcriptional silencing of the genomic region that has been methylated. During differentiation in the developing embryo whole regions of the genome will be methylated and therefore transcriptionally silenced. The DNA methylation patterns are maintained during cell division and future generations of that cell.

Analysing transcription on a global scale

For many years individual scientists would study the transcriptional regulation of their ‘favourite’ gene and so we gained an understanding of how individual genes were regulated in response to different development or environmental signals, for example the control of the lac operon in response to lactose and glucose. In the last 15 years, many techniques have been developed to allow us to study transcriptional control of genes within a cell. Using techniques such as ‘RNA-Seq’, we can isolate the total RNA from a cell and use high-throughput sequencing to catalogue the level of transcription of all genes. In the case of eukaryotes this will also show how they have been spliced. It is also possible to analyse the binding of transcription factors and study epigenetic changes within histone proteins across the genome using techniques such as ChIP-Seq. So, combining techniques such as RNA-Seq and ChIP-Seq we can determine when and where a protein factor is bound to DNA and study epigenetic changes in a particular cell type and the consequences in terms of gene transcription. In combination these techniques give a detailed picture of the factors that affect transcription; this has been used, for example to look at differences between cancer cells and normal cells from the same patient.

Transcription and disease

Transcription factors and promoters play major roles in health and disease, below are just a few examples to give an idea of their role in health and disease.

The transcription factor p53 is a tumour suppressor protein, it guards against cancer and some human cancers have mutations that knock out p53 function.

The drug Tamoxifen used in the treatment of breast cancer binds the oestrogen receptor inhibiting its function. The oestrogen receptor is a transcription factor that turns on the transcription of genes in response to oestrogen.

Rett Syndrome is a neurodevelopmental disorder that affects approximately 1 in 15000 female births. It is due to mutations in a transcription factor that would normally repress transcription of specific genes, the mutations lead to inappropriate transcription of these genes.

Cocaine use results in changes in expression of many genes, this can include epigenetic changes within genes involved in cognition and brain function. These epigenetic changes can be inherited and there is evidence that cocaine use by a father can result in epigenetic changes that result in male, but not female, offspring being cocaine resistant.

The key player in protein synthesis is the ribosome, a complex structure composed of RNA and proteins. The ribosome provides a framework that ensures that the mRNA and tRNA are correctly positioned enabling the deciphering of the genetic code. There are many other proteins that are important in protein synthesis; some of these are part of the ribosome and some are again correctly positioned by the framework of the ribosome. As we will see, the small subunit ribosomal RNA is a ribozyme; an RNA molecule with catalytic properties similar to those of enzymes. Ribosomal RNA can form a peptide bond between two amino acids.

Transfer RNA

The other nucleic acid that you need for protein synthesis is the tRNA. The tRNA molecule is single stranded and folds up into a characteristic structure by base pairing ( Figure 14 ). These act as adaptor molecules, each has an anticodon for a specific mRNA codon and each carries the amino acid specified by that codon. The anticodon has a complementary sequence to the codon on the mRNA.

( A ) Tertiary structure of the phenylalanine tRNA from yeast showing the anticodon (grey), the acceptor stem (violet) with the nucleotides CAA at the 3′ OH end (yellow). Image modified from ‘TRNA-Phe yeast’ Yikrazuul (licensed under CC BY-SA 3.0). ( B ) Clover leaf representation of the secondary structure of tRNA.

The enzymes which attach amino acids to tRNAs are called aminoacyl tRNA synthetases; they recognise a specific amino acid and the corresponding tRNA. The reaction also requires ATP, it is carried out in two steps:

In the first step the enzyme hydrolyses ATP releasing pyrophosphate (PP) and in the second it attaches the amino acid to the 3′ hydroxyl of the tRNA. Aminoacyl tRNA synthetase enzymes are highly specific, they recognise specific amino acids and will only attach them to the correct tRNA. This ensures correct coupling of amino acids and tRNA molecules which is just as important in ensuring the fidelity of protein synthesis as the matching of the anticodon to the codon by the ribosome. In addition this step is said to activate the aminoacyl tRNA as it not only produces the correct substrate for the ribosome but also provides much of the energy required for peptide bond formation during protein synthesis.

Structure of the ribosome

All living things contain ribosomes. The ribosomes in bacteria are slightly smaller than those found in eukaryotic cells ( Table 2 ) but the overall structure and the way in which they work are essentially the same. The 2009 Nobel Prize for Chemistry was awarded to three scientists, Ada Yonath, Thomas Steitz and Venkatraman Ramakrishnan, who used X-ray crystallography to solve the three-dimensional structure of the bacterial ribosome. The ribosome is composed of two subunits, the small subunit which reads the messenger RNA and the large subunit which forms the bonds between amino acids, adding them to the growing polypeptide chain. There are three important binding sites for tRNAs in the ribosome which are at the interface between the two subunits and only formed when the two subunits come together. These sites are shown on the image in Figure 15 , they are referred to as the acceptor or aminoacyl (A) site, the peptidyl (P) site where the peptide bond between amino acids is formed and the exit (E) site from which spent tRNAs leave the ribosome.

The structure of the ribosome of Thermus thermophilus showing the small subunit (green), large subunit (blue), mRNA (red) and three tRNAs in the acceptor, peptidyl and exit sites (yellow)

In ( A ) the new tRNA is delivered to the ribosome by elongation factor EF-Tu (purple). In ( B ) the amino acid on the incoming tRNA is brought close to the amino acid on the tRNA in the peptidly site to facilitate peptide bond formation (bright green) (Adapted from Goodsell 2010 , licensed under CC-BY-4.0 licence).

S stands for the Svedberg unit for sedimentation velocity.

In addition to the ribosome, the mRNA and tRNA, there are a number of small proteins that are not part of the structure of the ribosome, but are required for protein synthesis: initiation factors, elongation factors and termination factors. The importance of these factors is illustrated by the inherited condition Vanishing White Matter Disease (VWM). This serious neurodegenerative disease which results in lesions in the white matter in the brain is due to mutations in one of the initiation factors.

Protein synthesis

During protein synthesis the ribosome brings together the amino acid charged tRNA and the mRNA, the codon and anticodon are matched and the amino acids are joined together in the correct sequence. There are three phases to this process: initiation where the ribosome assembles on the mRNA, elongation where the triplet code is read and amino acids are added to the growing peptide chain and termination where protein synthesis stops.

A complex of proteins called the cap-binding complex bind to the 5′ cap of the mRNA ( Figure 10 ) in the nucleus. The mRNA is then exported to the cytoplasm where it recruits initiation factors, tRNA charged with a methionine and the small (40S) ribosomal subunit. Initiation factors also bind and the small subunit scans along the 5′ untranslated region of the mRNA until it encounters the first AUG start codon (Figure 16A). This is recognised by the anticodon codon of the initiator tRNA, the large subunit then docks to give the translation complex. The 80S ribosome with the tRNA charged with methionine at the P site is now ready to accept the next tRNA ( Figure 16 B).

( A ) During initiation, the mRNA recruits a tRNA charged with a methionine and the small ribosomal subunit, ( B ) the large subunit then docks to give the translation complex, ( C ) a tRNA with an amino acid attached enters the A site, ( D ) the peptide bond is formed between the amino acid in the P site and the one in the A site. The effect is that the growing peptide chain is transferred to the incoming aminoacyl tRNA in the A site leaving an empty tRNA in the P site. ( E ) Finally, everything moves along the mRNA by one codon in a process called translocation so the peptidyl tRNA with the growing peptide chain attached moves to the P site and the spent tRNA to the E site from where it leaves the ribosome. ( F ) When a stop codon is in the A site, a termination or release factor enters the A site, ( G ) the peptide is released from the ribosome and ( H ) the two subunits of the ribosome disassociate and are recycled.

With initiation complete, the mRNA is in the correct reading frame with the A site empty and the next codon exposed. In the elongation phase an aminoacyl tRNA, one charged with an amino acid, is brought to the ribosome in a complex with an elongation factor and enters the A site. If the anticodon it carries is complementary to the exposed codon it is correctly positioned in the acceptor site and GTP is hydrolysed on the elongation factor ( Figure 16 C). A peptide bond ( Figure 17 ) is then formed between the C terminus of the amino acid in the P site and the N terminus of the amino acid in the A site, this reaction is catalysed in the peptidyl transfer centre of the large subunit of the ribosome. The effect is that the growing peptide chain is transferred to the incoming aminoacyl tRNA in the A site leaving an empty or spent tRNA in the P site ( Figure 16 D). Finally, the peptidyl tRNA with the growing peptide chain attached moves to the P site. This step is called translocation and the energy being provided by hydrolysis of GTP by the elongation factor EF-G. The spent tRNA moves to the exit site from where it can leave the ribosome. The mRNA moves so that the next codon is exposed in the A site ( Figure 16 E) ready to accept a new aminoacyl-tRNA charged with another amino acid. During the elongation phase the ribosome cycles through this process, adding amino acids to the growing peptide chain until a stop codon is exposed in the A site. The new protein emerges from the ribosome through an exit tunnel in the large subunit.

Amino acids and peptide bonds

( A ) Amino acids consist of a carbon atom with an amine group (the N terminus), a carboxylic acid group (the C terminus) and a variable R group. The simplest R group is a methyl group giving the amino acid alanine. ( B ) When two amino acids are joined together a peptide bond is formed between the N terminus of one amino acid and the C terminus of another. This is a condensation reaction releasing one molecule of water.

Termination

The stop codon is not decoded by being recognised by an anticodon on a tRNA. Instead it is detected by proteins called termination or release factors. In eukaryotes there is a single release factor (RF1) that recognises all three stop codons enters the A site ( Figure 16 F). The ester bond linking the peptide chain to the tRNA in the P site is broken and the peptide is released from the ribosome ( Figure 16 G) The two subunits of the ribosome disassociate and are recycled ( Figure 16 H).

The structure and function of ribosomes are highly conserved with a large core of structurally conserved proteins and rRNAs found in both eukaryotic and prokaryotic ribosomes. However, there are some differences both in the rRNAs and in some of the additional proteins involved in translation ( Table 2 ). The elongation phase is highly conserved but there are important differences in how protein synthesis is initiated. Bacterial mRNAs have a specific sequence called the ribosome binding site or Shine–Dalgarno sequence. In order to ensure that the mRNA is correctly positioned in the ribosome the Shine–Dalgarno sequence binds to a complementary sequence of the 16S rRNA in the small subunit. In bacteria the initiator tRNA is charged with a modified amino acid N-Formylmethionine.

Differences between the structure of bacterial and eukaryotic ribosomes can be exploited by antibiotics which are selective in that they affect protein synthesis in bacteria but not in mammalian cells. Macrolide antibiotics like erythromycin, block the exit tunnel in the large subunit of bacterial ribosomes and halt protein synthesis. The exit tunnel in eukaryotic ribosomes is slightly narrower which means that eukaryotic ribosomes are not affected. Streptomycin, an important antibiotic in the treatment of tuberculosis binds to the 16S of bacterial ribosomes. This distorts the structure of the decoding site and results in misreading of the mRNA.

Polyribosome

Protein synthesis can proceed very quickly, particularly in rapidly growing cells or those that are differentiating. In bacteria between 15 and 20, new peptide bonds can be formed per second. In eukaryotes it is slower, more like five peptide bonds per second. A small human protein like insulin would take only 10 seconds to make whereas the largest human protein titin, which is found in human muscle cells, takes about an hour and a half per molecule. One of the mechanisms that ensures that protein synthesis is carried out efficiently is the polyribosome. As soon as one ribosome has started translation another ribosome binds to initiate synthesis of another protein copy. This gives rise to polyribosomes or polysomes which can be seen by electron microscopy. Recent cryo-EM images show that ribosomes can be arranged very closely on the mRNA with the mRNA entry and exit channels aligned to allow the smooth passage of mRNA between them ( Figure 18 ). Sometimes these polyribosomes can form circular structures so that, as soon as the ribosome has finished synthesis of one polypeptide it can rebind the same mRNA molecule and start synthesis of another copy of the protein.

The polyribosome

Cyro-electron micrograph reconstruction of eukaryotic polyribosome. Reprinted from ( Myasnikov 2014 ) by permission.

Closing remarks

The study of nucleic acids, from their first identification as the genetic material is littered with landmarks in molecular biosciences, many of them marked with Nobel Prizes. Since Watson and Crick proposed their structure of DNA our knowledge about DNA and how it works has expanded almost exponentially. The topics introduced in this article are important topics covered in all bioscience programmes; understanding them is key to all areas of biosciences from evolution and animal diversity to health and disease. Recent developments in the techniques that we can use to study DNA, often in living cells means that new and exciting developments in our understanding of the way nucleic acids work are occurring all the time. Given the scope of this article we have barely scratched the surface of the topic, however, the reader can find more detail from the articles in the bibliography below and even more detail from a few minutes searching on the internet.

The authors declare that there are no competing interests associated with the manuscript.

deoxyribonucleic acid

general transcription factor

origin of replication

ribonucleic acid

RNA-induced silencing

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Home — Essay Samples — Science — Protein — Protein Synthesis: Understanding the Process and its Importance

Protein Synthesis: Understanding The Process and Its Importance

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Published: Feb 7, 2024

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Table of contents

The process of protein synthesis, a. transcription, b. translation, the role of dna in protein synthesis, the role of rna in protein synthesis, regulation of protein synthesis, importance of protein synthesis.

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Here's How Much Protein You Should Eat After A Workout To Optimize Muscle Gain

W hether your goal is to have Dwayne Johnson's powerful legs or Jessica Biel's chiseled arms, you'll probably need to spend some time at the gym . Your workout might consist of 4 sets of 8 or a hard circuit with 50 reps to create those small tears in your muscles that will lead to muscle gain. However, no matter how hard you work out, you won't be able to add muscle without the right nutrition plan, starting with protein.

According to Medical News Today , your body continually breaks down muscle protein, so you need at least 0.8 grams of protein per kilogram of body weight each day just to prevent muscle loss . To gain muscle, you'll need to add more protein to your overall nutrition plan, with the optimal amount falling in the range of 1.2 to 1.6 grams of protein per kilogram of body weight. For a 150-pound person, that's between 82 and 109 grams of protein a day.

However, you don't want to eat all that protein in one sitting, especially after your workout. According to a 2017 article in the Journal of the International Society of Sports Nutrition, you should consume about 20 to 40 grams of high-quality protein within two hours after your workout to optimize muscle gain.

Read more: When You Only Do Cardio, This Is What Happens To Your Body

Post-Workout Nutrition Plays A Key Role In Gaining Muscle

How much protein you'll need after your workout depends on your body weight. The International Society of Sports Nutrition article suggests 0.25 to 0.40 grams of protein for each kilogram of body weight. If you weigh 150 pounds, that's 17 to 27 grams of protein to stimulate muscle protein synthesis.

While a hard muscle-building workout requires protein for muscle synthesis, you'll also need to add some carbs to help your body absorb protein and replenish lost muscle glycogen. Your body will need 0.8 grams of carbohydrates for each kilogram of body weight, which is about 54 grams of carbs for someone weighing 150 pounds.

You can easily reach these goals with a blueberry banana smoothie. A cup of blueberries, a medium banana, and a scoop of whey protein give you 51 grams of carbohydrates and 26 grams of protein (depending on your brand of protein powder). According to a 2013 article in the Nestle Nutrition Institute Workshop Series, whey protein is best after a workout because it's absorbed by your body more rapidly to get to work on building that muscle.

Protein Throughout Your Day

Although it might be tempting to add more protein to your post-workout meal, the International Society of Sports Nutrition article says it's best to spread out your protein intake throughout the day, preferably every three hours or so. Your body is still synthesizing protein between three and five hours after exercise. Adding about 30 or 40 grams of casein protein in the evening will stoke your metabolism and the recovery process.

Protein sources such as meat, chicken, eggs, seafood, and dairy have the nine essential amino acids that you need from food. Plant-based proteins like beans, peas, lentils, and nuts don't have sufficient amounts of all these amino acids on their own (via Medical News Today). Therefore, vegans and vegetarians should include a variety of plant-based protein sources to meet their amino acid needs to build muscle.

You'll also still need plenty of carbs to sustain your energy and help keep your body fueled for your next workout. Even bodybuilders preparing for a competition will take in between 55 and 60% of their total calories from carbohydrates, according to a 2004 review in Sports Medicine.

Read the original article on Health Digest .