wobble hypothesis

The Wobble Hypothesis: Importance and Examples

The “wobble hypothesis” refers to a concept in molecular biology that explains the degeneracy of codons. 

So, what are codons? Codons are sets of three nucleotides in mRNA (messenger RNA) that correspond to specific amino acids. 64 possible codons codes for the 20 standard amino acids used in protein synthesis. 

Since there are only 20 amino acids and 64 possible codons, multiple codons may code for a single amino acid during protein synthesis. In molecular biology, this redundancy or multiplicity of codons is termed degeneracy.

The wobble hypothesis or wobble theory, proposed by Francis Crick in 1966, suggests that the third base of a codon can sometimes be flexible or “wobble.” The wobbling or flexibility allows for non-standard base pairing between the mRNA codon and the tRNA (t RNA) anticodon during translation.

The first two nucleotides of the codon typically adhere to strict base-pairing rules. Still, the third position may tolerate mismatches, allowing for variations such as G-U (guanine-uracil) pairing or other non-standard interactions.

This hypothesis helps explain how a relatively limited number of tRNA molecules can recognize and bind to multiple codons for the same amino acid, facilitating efficient and accurate protein synthesis. Experimental evidence has supported the wobble hypothesis, a fundamental concept in understanding the genetic code and translation machinery.\

Table of Contents

Crick’s wobble hypothesis states that the base at the 5′ end of the anticodon does not confine spatially as the other two bases, which allows the development of hydrogen bonds with other bases present at the 3′ end of a codon. The wobble hypothesis outlines several key points:

• Degeneracy of the Genetic Code: The genetic code degenerates, meaning multiple codons can code for the same amino acid. For example, six codons, UUA, UUG, CUU, CUC, CUA, and CUG, code the amino acid leucine.
• Flexibility in Codon-Anticodon Interactions: The wobble hypothesis suggests that the base pairing between the mRNA codon’s third nucleotide and the tRNA anticodon’s corresponding nucleotide is flexible. Instead, it allows for some flexibility or “wobble” in the pairing.
• Non-Standard Base Pairing: The third position of the codon-anticodon interaction can tolerate non-standard base pairs, such as G-U (guanine-uracil) pairing or other non-Watson-Crick interactions. For example, a tRNA with the anticodon 3′-CCU-5′ can recognize the codons CGU, CGC, and CGA, where the third position allows for wobble pairing.

Importance of Wobble Hypothesis

The wobble hypothesis is essential in molecular biology for several reasons:

• Efficient Translation : The wobble hypothesis explains how fewer tRNA molecules can recognize multiple codons coding for the same amino acid. This reduces the number of tRNA species required for protein synthesis, streamlining the translation process and making it more efficient.
• Error Reduction : By allowing for flexibility in base pairing at the third position of the codon-anticodon interaction, the wobble hypothesis helps reduce the impact of errors or mutations in the genetic code. Even if a mutation occurs in the third position of a codon, it may not necessarily result in a change in the protein’s amino acid sequence, thereby minimizing errors in protein synthesis.
• Evolutionary Conservation : The wobble hypothesis is evolutionarily conserved across species, indicating its fundamental importance in translation. This conservation suggests that the wobble base pairing mechanism provides an evolutionary advantage by allowing for greater adaptability and efficiency in protein synthesis.
• Understanding Genetic Code Variability : The wobble hypothesis helps us understand the variability in the genetic code, where multiple codons can code for the same amino acid. This variability provides flexibility and redundancy in the genetic code. This allows for robustness and adaptability in the face of genetic mutations and environmental changes.
• Biotechnological Applications : Understanding the wobble hypothesis is crucial in biotechnology and genetic engineering applications. For example, it informs the design of synthetic genes and optimization of codon usage to enhance protein expression in heterologous expression systems.

Overall, the wobble hypothesis plays a fundamental role in understanding protein synthesis and the genetic code, with implications for various aspects of molecular biology, genetics, and biotechnology.

Examples of Wobble Hypothesis

Here are some examples of the wobble hypothesis in action:

• Arginine: The amino acid arginine is coded by six different codons: CGU, CGC, CGA, CGG, AGA, and AGG. However, no six different tRNA molecules correspond to each of these codons. Instead, one tRNA molecule with the anticodon 3′-CCU-5′ can recognize the codons CGU, CGC, and CGA (where the third nucleotide is flexible), thanks to wobble base pairing.
• Leucine: Leucine is another example of wobble base pairing. The codons UUA, UUG, CUU, CUC, CUA, and CUG are all codes for leucine. However, the tRNA molecule with the anticodon 3′-AAG-5′ can recognize UUA and UUG codons due to wobble base pairing at the third position.
• Serine: Serine is encoded by six codons: UCU, UCC, UCA, UCG, AGU, and AGC. Due to wobble base pairing, the tRNA molecule with the anticodon 3′-AGU-5′ can recognize both AGU and AGC codons.
• Isoleucine: The codons AUU, AUC, and AUA all code for isoleucine. The tRNA molecule with the anticodon 3′-IAU-5′ (where “I” represents inosine, a modified nucleotide capable of wobble base pairing) can recognize all three codons through wobble interactions.

These examples illustrate how the wobble hypothesis allows for flexibility in the genetic code, enabling fewer tRNA molecules to recognize multiple codons and facilitating efficient protein synthesis.

Limitation of Wobble Hypothesis

While the wobble hypothesis provides a valuable framework for understanding how the genetic code is flexible and the efficiency of translation, it also has some limitations and considerations:

• Context-dependence : The wobble hypothesis primarily applies to the standard codon-anticodon interactions during translation. However, non-standard base pairing beyond the wobble hypothesis may occur in certain contexts or under specific conditions. For example, modified nucleotides in tRNA or mRNA can influence base pairing interactions in ways that go beyond traditional wobble pairing rules.
• Accuracy and Specificity : While wobble base pairing can contribute to the recognition of multiple codons by a single tRNA molecule, it may also lead to potential errors during translation. The flexibility in the third position of the codon-anticodon interaction could allow non-standard base pairs to form. This can potentially lead to misinterpretation of the genetic code and errors in protein synthesis.
• Influence of Structural Constraints : The wobble hypothesis primarily focuses on the base pairing interactions between codons and anticodons. However, other factors such as tRNA structure, modifications, and interactions with the ribosome also influence the accuracy and efficiency of translation. These factors may impose additional constraints or considerations beyond the wobble hypothesis.
• Evolutionary Variability : While the wobble hypothesis explains a general trend in codon-anticodon recognition, there can be variations in wobble base pairing preferences across species or even within different tissues or cellular conditions. Evolutionary pressures, genetic variations, and differences in tRNA modifications can influence the extent and specificity of wobble interactions.
• Complexity of Codon Usage : The relationship between codon usage bias, tRNA abundance, and wobble interactions is complex and can vary between organisms and genes. While wobble base pairing contributes to codon redundancy and efficient translation, other factors such as codon optimality, mRNA secondary structure, and ribosome kinetics influence translation efficiency and protein expression levels.
• Crick F. H. (1966). Codon–anticodon pairing: the wobble hypothesis. Journal of molecular biology , 19 (2), 548–555. https://doi.org/10.1016/s0022-2836(66)80022-0  
• Mangang, S. U., & Lyngdoh, R. H. (2001). Wobble base-pairing in codon-anticodon interactions: a theoretical modelling study. Indian journal of biochemistry & biophysics , 38 (1-2), 115–119.
• Verma, P. S., & Agarwal, V. K. (2019). Cell Biology, genetics, Molecular Biology, evolution and ecology (25th ed.). S. Chand and Company Limited.

Ashma Shrestha

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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• Published: 21 January 2016

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

• Alexey Rozov 1 , 2   na1 ,
• Natalia Demeshkina 1 , 2   na1 ,
• Iskander Khusainov 1 , 2 , 3 ,
• Eric Westhof 4 ,
• Marat Yusupov 1 , 2 &
• Gulnara Yusupova 1 , 2  

Nature Communications volume  7 , Article number:  10457 ( 2016 ) Cite this article

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• Molecular biophysics
• Structural biology

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNA Lys UUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm 5 s 2 U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

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Introduction.

More than a hundred of posttranscriptional RNA modifications identified today 1 were shown to play diverse and indispensable roles in gene regulation in all domains of life 2 . Modifications of RNA are carried out by complex cellular pathways, which involve countless protein enzymes and catalytic RNA–protein complexes, which primarily target tRNAs and, to a lesser extent, ribosomal RNA and mRNAs 1 . The observed trends suggest that many modification motifs and their sequence locations are conserved throughout Bacteria, Archaea and Eukarya; however, some kingdom-specific differences are documented as well.

Among all modifications found, those of tRNA are the most abundant and studied classes of modifications 1 , 3 , 4 , 5 . Ninety-three tRNA modifications described today represent an astonishing library of chemically diverse structures 5 each of which influences in a unique way three-dimensional integrity of the tRNA and specifies its physicochemical properties 6 , 7 . The most elaborate tRNA modifications are located in the anticodon loop, which comprises the anticodon triplet necessary for pairing with mRNA codons. The anticodon loops of almost all tRNAs contain several modified nucleotides. Among these, the most important are nucleotides in the positions 34 and 37. The nucleotide in position 34 (so-called ‘wobble’ position) pairs with the third mRNA codon base in the aminoacyl-tRNA binding site (A-site) during decoding 4 , 8 . The nucleotide in the position 37 is adjacent to the 3′-side of the anticodon.

From the moment of their discoveries, modifications in tRNA anticodon loops were demonstrated to be crucial for proper mRNA decoding and fine-tuning of the process 9 . In particular, modifications of tRNA position 34 were implied to increase tRNA capacities to decode multiple mRNA codons differing by the third nucleoside (synonymous codons), hence explaining how the degenerate genetic code is translated 4 , 10 , 11 . It was shown also that anticodon modifications enhance recognition by corresponding aminoacyl-tRNA synthetases 12 , 13 and serve as a preventing measure of frame shifting during translocation 14 , 15 . Very recently, the tRNA modifications were also credited a role in connecting translation, metabolism and stress response in bacteria and eukaryotes 2 , 16 . For example, in humans the deregulation of RNA modification pathways was shown to be linked to the type two diabetes and several mitochondrial diseases 16 .

Except for methionine and tryptophan, all amino acids are encoded by more than one codon, because the genetic code is redundant 17 , 18 with 61 codons encoding 20 amino acids. In contrast to eukaryotes, where synonymous codons AAA and AAG are read by three isoacceptor lysine tRNA Lys , half of all bacteria have only one isoacceptor tRNA Lys UUU that decodes these two codons into amphipathic amino acid lysine 9 , 19 , 20 . To discriminate against pyrimidine-ending codons AAC and AAU encoding asparagine, the E. coli tRNA Lys UUU contains one of the most complex modifications 5-methylaminomethyl-2-thiouridine (S, mnm 5 s 2 U) at the wobble anticodon position 34 ( Fig. 1a ). The mnm 5 s 2 modification is a result of a sophisticated pathway that includes many enzymes responsible for thiolation and attachment of a methylaminomethyl group 21 . Another prominent feature of E. coli tRNA Lys SUU is N6-threonylcarbamoyladenosine (t 6 A) at the 37th position of its anticodon loop ( Fig. 1a ). The t 6 A modification is one of the most ubiquitous and conserved, and is known to be critical for recognition of codons starting with adenosine. This is one of the rare modifications universally conserved throughout different kingdoms of life 1 . In addition to S34 and t 6 A37, E. coli tRNA Lys SUU anticodon loop bears the third modification, pseudouridine at position 39 ( Fig. 1a ).

( a ) Secondary structure of E. coli tRNA Lys SUU . Major domains and modifications of tRNA are indicated; chemical formulas of hypermodified nucleobases at positions 34 and 37 are given together with corresponding abbreviations. ( b ) Side view of the 70S ribosome complex with three tRNAs bound at the A- (red), P- (blue) and exit (green) binding sites. Helix 69 of the large subunit is in magenta. The frame designates the decoding centre with the bound anticodon-stem loop of tRNA Lys SUU and the close-up view on the codon–anticodon duplex and major nucleotides of the decoding centre (G530 from 16S rRNA is not shown) including A1913 from 23S rRNA. ( c ) Schemes of codon–anticodon duplexes in the decoding centre of the 70S ribosome complexes modelled in the study. The complexes are numbered in accordance with description in the main text.

Nuclear magnetic resonance studies of unmodified, partially and fully modified anticodon stem loops (ASLs) of E.coli tRNA Lys SUU demonstrated that mnm 5 s 2 U and t 6 A modifications remodel an otherwise dynamic loop to canonical open U-turn structure to perfectly adapt in the ribosomal decoding centre 6 , 22 . First, X-ray studies of the partial decoding system 23 , where crystals of the isolated small ribosomal 30S subunit were soaked with synthetic ASL of E.coli tRNA Lys UUU and hexaribonucleotides as mRNA analogues, shed some light on possible roles of modifications in decoding 24 , 25 . It was suggested that t 6 A37 enhances the codon–anticodon stability via cross-strand stacking interaction with the first codon nucleotide, whereas partially modified mnm 5 U34 lacking 2-thio group was implicated in an alternate mnm 5 U34·G base-pairing interactions via a bifurcated hydrogen bond 25 . A similar model of the bacterial 30S subunit with ASL of human tRNA 3   Lys UUU , which has identical anticodon loop sequence with E. coli tRNA Lys SUU but carries ms 2 t 6 A37 (2-methylthio-N6-threonylcarbamoyladenosine) and mcm 5 s 2 U34 (5-methoxycarbonylmethyl-2-thiouridine) modifications, revealed Watson–Crick-like geometry of mcm 5 s 2 U34·G base-pairing interactions at the wobble codon–anticodon position 26 . Both of these works and numerous other genetic, biophysical and biochemical findings indicated that each group in a modified nucleotide improves thermodynamic properties of tRNA and serves to augment specific codon–anticodon interactions during decoding.

Recent crystallographic studies of the 30S ribosomal subunit or complete 70S ribosome complexes with different ASLs 23 , 24 , 25 , 26 , 27 or tRNAs 28 , 29 , 30 , 31 , 32 , 33 and mRNAs describe three major classes of preferred geometry at the wobble position of a codon–anticodon minihelix. The first class consists of canonical Watson–Crick purine–pyrimidine A·U (or U·A) and C·G (or G·C) pairs 29 , 30 , 33 , and also includes Watson–Crick-like pairs. It was suggested that the latter closely resemble canonical geometry via stabilization of an enol tautomer by the wobble modifications (for example, above described mcm 5 s 2 U34·G) 26 , 27 . The second class consists of standard wobble pair G34·U originally predicted by Crick 8 . In this pair, the pyrimidine is displaced towards the major groove of the codon–anticodon minihelix; however, the distance between the ribose C1′ atoms remains very close to 10.5 Å, the average value for a standard Watson–Crick pair 28 , 29 . The third class includes I·G or I·A pairs, where I (inosine) is present at position 34 of some tRNAs 1 , 34 . These purine–purine pairs are unusually wide with a C1′–C1′ distance of 12.3 Å (refs 24 , 32 ).

In the current study, we describe six X-ray structures of physiologically relevant complexes of the complete 70S ribosome primed with long mRNAs that contain full-length native E. coli tRNA fMet in the peptidyl-tRNA-binding site (P-site) and tRNA Lys SUU bound to cognate or near-cognate codons in the A-site. We identified an unprecedented base-pairing interaction at the wobble position of the codon–anticodon duplex in the decoding centre that broadens the present family of ‘wobble geometries’. This base pair, which involves a hypermodified S34 of E. coli tRNA Lys SUU and codon guanosine, represents a ‘wobble’ (G34·U) with the U moved towards the minor instead of the major groove that is much less isosteric to its flipped form than usual wobble G·U pair.

The ribosome structures we are describing in this work deepen the understanding of the tRNA discrimination mechanism on the ribosome. We demonstrate how tRNA Lys SUU discriminates between cognate codons AAA and AAG, and the near-cognate stop codon UAA (ochre codon) or the isoleucine codon AUA, with which it forms pyrimidine–pyrimidine U·U mismatches. Together with our earlier structures of the 70S ribosome with various mismatches in the codon–anticodon duplex 29 , 30 , the present models expand our library of various states of the 70S decoding centre. The present evidence further strengthens our proposition that the steric complementarity is predominant over the number of hydrogen bonds 35 between the decoding centre and the codon–anticodon duplex, and hence plays the crucial discriminatory role during decoding.

The modifications of lysine tRNA in the 70S decoding centre

In this work as in our previous studies 28 , 29 , 30 , we employed the full Thermus thermophilus 70S ribosome co-crystallized with long synthetic mRNAs and natural tRNAs. These complexes model cognate or near-cognate states of the decoding centre at the proofreading step of the tRNA selection process ( Fig. 1b ). We determined six X-ray structures of the 70S ribosome programmed by 30-nucleotide-long mRNAs with AUG codon and E. coli tRNA fMet in the P-site and the A-site occupied by tRNA Lys SUU bound to its cognate codon AAA or AAG, or near-cognate stop codon UAA and isoleucine codon AUA ( Fig. 1c and Table 1 ).

The electron density maps of the two cognate complexes ( Fig. 1c , complexes 1 and 2) possess sufficient level of detail to discern structural features of the tRNA anticodon loops that can be attributed to the influence of modifications mnm 5 s 2 U and t 6 A ( Fig. 2 ). The amino group of the modified nucleotide S34 forms a hydrogen bond with 2′-OH of nucleotide U33 ( Fig. 2a ), thus altering the U-turn structure and stabilizing it, as was shown before for isolated ASLs 6 . The thio group of the same nucleotide is known to stabilize 3′-endo conformation of the ribose favourable for base-pairing interactions 36 , influencing the codon–anticodon helix stability. We observed a feature, characteristic of 2-thiouridine 37 as well, namely the S2(S34)-N1(U35) ‘stacking’ interaction with the subsequent nucleotide U35 ( Fig. 2a ). This interaction affects the relative positioning of the S34 and U35 nucleotides, and hence the shape and stability of the codon–anticodon duplex.

( a ) The S34 interactions: stabilization of the U-turn structure via anchoring of the mnm 5 group and stacking of the thio group with the N1 atom of U35 shown in an alternative top-view orientation on the right. Nucleotide 37 is omitted for clarity of representation. ( b ) The invariant A1913 in Helix 69 of 23S rRNA of the large ribosomal subunit (thick magenta line) constrains position of the t 6 A37 ribose by conserved hydrogen bond interactions; van der Waals surfaces show that A1913 also defines conformation of the anticodon loop from the 3′-side of t 6 A37 at position 38. ( c ) Cross-strand stacking of t 6 A37 with the first nucleotide of the A-site bound codon (position (+4)). In a and c , 2F o − F c electron density maps corresponding to S34 and t 6 A37 are contoured at 1.0 σ .

An influence of the large 50S ribosomal subunit on the tRNA constraints during decoding remained underestimated for a long time, because the first models of decoding were based on the structures of the isolated small ribosomal subunit 23 , 35 . On the 70S ribosome, the conserved helix 69 of the 50S subunit, which is pivotal for many functions of the ribosome, directly contacts the sugar moiety of the tRNA nucleotide at position 37 (refs 31 , 38 and Fig. 2b ). Most probably, this contact is important for proper positioning and conformational stabilization of the anticodon loop. In addition, t 6 A37 forms cross-strand stacking with the first nucleotide of the mRNA codon in the A-site ( Fig. 2c ). Similar stacking interactions were described in the early models of the 30S subunit whose crystals were soaked with the tRNA Lys UUU ASL carrying t 6 A37 and mnm 5 U34 modifications 25 . However, the comparison of our present structure with this model revealed a considerable shift of 1 Å in the position of the t 6 A37 nucleotide pointing to a specific role of the large 50S subunit in restraining the anticodon loop of the A-site bound tRNA ( Supplementary Fig. 1 ). The position of t 6 A37 over the A·U pair itself ( Fig. 2c ), as observed in our structures, corresponds well with the main function of this modification in stabilizing the weak A·U base-pairing interactions and preventing mRNA slippage during translocation as well.

Hypermodified uridine forms a unique base pair

The capacity of E. coli tRNA Lys SUU to read both codons AAA and AAG ending with purines implies that the hypermodified uridine S34 at the first anticodon position is involved in a dual mode of base-pairing interactions with adenosine and guanosine. In general, in bacteria the AAA codon is used approximately three times more often than the AAG codon 39 . However, it was estimated that when the next codon after the one encoding lysine starts with cytidine the AAG codon becomes preferred 40 .

Our first structure with E. coli tRNA Lys SUU bound to its cognate AAA codon showed that hypermodified uridine S34 formed a distorted Watson–Crick base pair with the opposing adenosine with the standard C1′–C1′ distance of 10.6 Å ( Fig. 3a ). The slight positional deviation of the uracil ring from its standard position in the Watson–Crick A·U pair, caused by the interactions of the modifications with neighbouring nucleotides U33 and U35 ( Fig. 2a ), resulted in weakening of interaction between the codon adenosine and S34. On the other hand, the very same interactions tend to strengthen the codon–anticodon duplex as a whole by adjustment of the shape of tRNA U-turn 6 ( Fig. 2a ).

( a ) The S34·A(+6) pair. Left, hydrogen bonds are indicated (Å); right, van der Waals surfaces with the corresponding C1′–C1′ distance and glycosidic angles ( λ ). ( b ) The S34·G(+6) pair; indicated parameters as in a ; possible hydrogen bonds are marked by red dashes. In a and b , the sugar moiety of codon nucleosides is coordinated by magnesium ion (shown in magenta) together with small subunit elements (Pro 48 of S12 and C518 with G530 in 16S rRNA); coordination distances show that the position of the wobble nucleotide in the codon can slightly adjust depending on the type of pairing interactions. The anticodon nucleotide is weakly restricted by C1054 in 16S rRNA via lone pair–aromatic interactions. The lower panel displays the standard wobble G34·U(+6) pair with specified parameters as in a and b ; see PDB code: 3H8I. ( c ) Superposition of S34·A(+6) and S34·G(+6) pairs by the mRNA codons (left) or by the tRNA Lys SUU anticodon (right) shows relative extent of the guanosine and modified uracil displacement in the S34·G(+6) pair; the displacement is estimated by angstroms and marked by red arrows. In a and b 2F o − F c electron density maps are contoured at 1.5 σ .

The model of tRNA Lys SUU bound to its second cognate codon AAG demonstrated an unprecedented and striking base-pairing geometry ( Fig. 3b ). The new S34·G(+6) pair is characterized by the larger C1′–C1′ distance of 11.5 Å that exceeds a corresponding distance in a standard Watson–Crick pair by 1 Å. Yet, interatomic distances between Watson–Crick edges within S34·G(+6) imply the existence of two hydrogen bonds between carbonyl oxygen and N3 atom of modified uracil, and N3 atom and amino group of guanosine, respectively ( Fig. 3b ).

One of the hypotheses that could explain observed pairing interactions suggested that under physiological conditions a significant fraction of mnm 5 s 2 U is present in a zwitterionic form ( Fig. 4a ) 41 . This is due to the increased acidity of the N3 proton by the inductive effect of the protonated methylaminomethyl group, as was predicted based on the theoretical estimate of the p K a (ref. 41 ). In its neutral form, the modified uracil forms two hydrogen bonds same as in the case for a standard Watson–Crick U·A pair and as we observed for the mnm 5 s 2 U34·A(+6) pair ( Fig. 4b ). In its deprotonated form, the N3 atom of the uracil becomes a proton acceptor and can form a hydrogen bond with the amino group of guanosine, while the uracil carboxyl group is engaged in another hydrogen bond with the guanosine N3 atom ( Fig. 4c ). Although less probable, the observed unusual pattern of hydrogen bonds between the modified uridine and guanosine can also be rationalized by existence of either rare tautomeric states ( Fig. 4d,e ) or an alternative zwitterionic state ( Fig. 4f ) of the modified uridine.

( a ) Equilibrium between two forms of mnm 5 s 2 U 41 . ( b ) Interactions with adenosine of the neutral form of mnm 5 s 2 U. ( c ) Theoretically predicted base pair with guanosine of zwitterionic form of mnm 5 s 2 U carrying negative charge on sulfur atom. ( d , e ) Two alternative pairing interactions with guanosine and enol tautomeric forms of mnm 5 s 2 U. ( f ) Base-pairing interactions with guanosine of a possible zwitterionic form of mnm 5 s 2 U.

It is worth to underline here that in contrast to the first two codon–anticodon positions, which are tightly restricted by the decoding centre 29 , 30 , the ‘wobble’ pair is not very firmly stabilized. The codon nucleotide is held in place only by indirect interactions with G530, C518 and S12 through a Mg 2+ ion ( Fig. 3a,b ) and the O4' of the first anticodon nucleotide forms weak off-centre lone pair–π interaction with the nucleobase of C1054 in 16S rRNA ( Supplementary Fig. 2 ) 42 . However, the fact that observed S34·G(+6) pair is distorted from the standard ‘wobble’ geometry suggests that the aforementioned indirect restraints and interactions of the modification groups provide some restraints to control geometry of the third base pair.

Pyrimidine–pyrimidine mismatch in the 70S decoding centre

The translation of genes into proteins is an error-prone process with the average frequencies of mistranslation 10 −3 –10 −5 (ref. 43 ). We have recently published first structural rationales for the phenomenon of translational infidelity 30 . We demonstrated that because the G·U mismatches can mimic the form of a canonical Watson–Crick pair via tautomerization or ionization, these type of mismatches become accepted in the decoding centre, which restricts geometries of allowed pairs to canonical interactions. At the same time, our models with the A·A and C·A mismatches at the first two positions of the codon–anticodon duplex suggested that these pairs would be efficiently discriminated against because of (i) steric clashes within a mispair or of a mispair with the tight decoding centre itself, or (ii) because of absence of stable pairing interactions between pairing nucleotides 29 , 30 .

In the current study, we asked an ensuing question of what is the structural basis for discrimination against the pyrimidine–pyrimidine U·U pair, which represents a low-probability mistake during translation 44 , 45 , 46 . Thus, we determined structures of complexes 3 and 4 ( Fig. 1c ) where the SUU anticodon of tRNA Lys SUU formed a U·U mismatch either with the first or the second codon position. As was anticipated, the nucleotides, critical for decoding A1493 and A1492/G530 of 16S rRNA 47 , stabilized the U·U mismatch at the first and the second codon–anticodon positions via A-minor groove interactions ( Fig. 5a ). These results substantiated our expanded mechanism of decoding that provides structural basis for discrimination in favour of correct tRNAs and against incorrect tRNAs, and describes identical rearrangements of the decoding centre on binding of cognate or near-cognate tRNA 29 , 30 . In both complexes, the interatomic distances between the Watson–Crick edges of opposing uracils exceeded 3.4 Å, implying weak electrostatic interactions ( Fig. 5b,c ). A hypothetical U·U pair possible through a shift in the keto-enol equilibrium would require a typical distance of 3.0–3.1 Å between uracils for hydrogen bonds to occur ( Fig. 5d ). However, positions of uracils in both models make this scenario unlikely ( Fig. 5b,c ). To put it simply, the restraints on the sugar-phosphate backbones of the codon–anticodon helix imposed by the decoding centre are strong enough to prevent the uracils to come close enough to interact strongly via hydrogen bonds ( Supplementary Fig. 3 ).

( a ) Critical A1492 and A1492/G530 of 16S rRNA stabilize the sugar phosphate backbones of the U·U mismatch at the first (left) and second (right) positions of the codon–anticodon duplex by A-minor groove interactions as is the case for any canonical Watson–Crick pair 29 , 30 . 2 F o − F c electron density maps are contoured at 1.5 σ . ( b , c ) Geometries of the U·U mismatch at the first ( b ) and second ( c ) positions of the codon anticodon duplex; van der Waals surfaces (left) together with interatomic distances (right) are presented. In b , the table describes standard categories of hydrogen bonds. ( d ) Formation of a strong U·U pair would necessitate a shift in the keto-enol equilibrium from abundant keto form (left panel) to a rare enol form (red frame) and the 3-Å distance between Watson–Crick surfaces of opposing uridines (right panel). ( e ) Conformation of the wobble S34·A(+6) pair in the complex 3 (see Fig. 1c ). The absence of two hydrogen bonds expected for the mnm 5 s 2 U·A pair can reflect (i) a deformation of the codon–anticodon minihelix induced by the mismatch and (ii) the specific influence of the tRNA modification at position 34 known to counteract misreading of the genetic code. ( f ) The near-cognate duplex composed of the tRNA Lys SUU anticodon and the ochre stop codon is significantly weakened compared with the cognate version on the AAA codon with the full set of canonical Watson–Crick interactions. The described weakening of the near-cognate duplex would imply dissociation of tRNA Lys SUU from the ribosome or, in other terms, rejection.

In complex 3 with the stop codon UAA and the first U·U mismatch, the codon–anticodon duplex was additionally weakened at the 3′-end where the S34·A(+6) pair was slightly deformed ( Fig. 5e ). As a result, only three strong hydrogen bonds were formed between codon and the anticodon compared with the cognate version with six hydrogen bonds ( Fig. 5f ). These results demonstrated why tRNA Lys SUU normally does not read the ochre codon. Two additional structures of the near-cognate complexes 3 and 4 ( Fig. 1c ) solved in the presence of antibiotic paromomycin, which stimulates miscoding, supported our previous conclusion of the antibiotic mechanism of action 48 . In both cases, paromomycin stabilized A1492 and A1493 in the ‘out’ from the interior of helix 44 positions, hence stimulating A-minor groove interactions with the first two nucleotides of the A-codon. In addition, binding of the antibiotic led to a positional shift of the A1493 phosphate, resulting in partial alleviation of the restrictive decoding centre from the side of the mRNA codon. Finally, in the ribosome structures with paromomycin we observed a displacement of helix 69 of the large subunit towards the D-stem of tRNA Lys SUU that, most probably, enhanced stabilization of a near-cognate substrate on the ribosome.

It is important to mention that uracils in a mismatch at the first codon–anticodon position were closer to each other than at the second position ( Fig. 5b,c ). In the light of kinetic description of the tRNA selection process on the ribosome, it can be interpreted as the second codon–anticodon position being more controlled than the first one 43 . Accordingly, kinetic evaluations of codon readings by tRNA Lys UUU in bacteria assigned the highest accuracy values for the second position in the codon–anticodon duplex 45 . Thus, it is important to indicate here that despite of the fact that we could crystallize described near-cognate states of the ribosome at the proofreading step, in solution these complexes would be prone for dissociation because of a prominent lack of pairing codon–anticodon interactions.

Fifty years ago, Francis Crick suggested some rules for translation of the genetic code on the ribosome and postulated the wobble hypothesis that gave first explanations to degeneracy of the code 8 , 18 . It was predicted that the first two positions of the codon would pair with the anticodon using the standard base pairs, while in the base pairing of the third codon base ‘there is a certain amount of play, or wobble, such that more than one position of pairing is possible’. First examples of foretold wobble non-standard pairs included G·U, U·G, I·A, I·C and I·U pairs, where the nucleoside on the left designates position 34 in tRNA 8 . Since those times, a titanic work on deciphering the genetic code resulted into a simple textbook chart where most of the codons are two- or fourfold degenerate, meaning that one amino acid can be coded by two or four codons differing by the third base. Further identification of tRNA modifications and especially those at the first anticodon ‘wobble’ position 34 elaborated more on the phenomenon of degeneracy. The ‘modified wobble hypothesis’ suggested that specific tRNA base modifications evolved to discriminate particular codons—expanding and facilitating an ability of tRNA to read more than one codon in some cases and preventing misreading in other cases 4 .

In the present study, we describe a new type of a base pair at the third wobble position of a codon–anticodon duplex in the 70S ribosome decoding centre. Our structures demonstrate that the reversed ‘wobble’ pair S34·G(+6) adopts its own geometry, different from the standard G34·U(+6) pair 8 at the third codon–anticodon position ( Fig. 3b ). This is possibly due to both certain restraints put on the third base pair by the decoding centre of the 70S ribosome and modifications on the nucleotide S34 of tRNA Lys SUU , shaping the codon–anticodon helix and the ASL of the tRNA (see Results). In spite of the fact that S34·G(+6) is not isosteric to the standard wobble pair, there is a certain similarity of the overall shape between these two pairs.

Both pairs are characterized by displacement of the anticodon nucleotide—S34 in the S34·G(+6) pair and G in the G34·U(+6) pair—towards the minor groove of the codon–anticodon minihelix. This is achievable because of the apical location of the nucleotide in the U-turn structure of the tRNA anticodon loop 49 . On the codon side of the pairs there is another tendency of shifting; however, in this case it is towards the major groove of the minihelix. Results reported in this study particularly show this displacement ( Fig. 3c right). Hence, a term ‘wobble’ can be also applied to the capacity of the third codon nucleotide to adjust its position because of difference in strength and type of restraints imposed by the decoding centre on the third base pair as compared with the first and second base pairs ( Fig. 3a,b ).

Uridine at the wobble positions of various tRNAs is almost always modified in bacteria and eukaryotes 1 , 5 . In many cases, tRNAs with the modified uridines read two codons ending with purine A or G and, in some rare cases, modifications help to recognize all four nucleotides A, G, C and U at the third codon position 50 . Previously, it was shown that preferential form of the third wobble pair with a fully modified uridine approached a standard Watson–Crick-like geometry if this uridine was paired with guanosine 26 , 27 ( Supplementary Fig. 4 ). Thus, in the partial and heterologous model of the isolated bacterial 30S ribosomal subunit, whose crystals were soaked with an ASL of human tRNA 3   Lys UUU , the modified mcm 5 s 2 U34 formed C·G-like mcm 5 s 2 U34·G pair 26 . In this pair, the uridine base was shifted towards the major groove when compared with geometry of the mnm 5 s 2 U·G(+6) pair in our model ( Supplementary Fig. 4b left). In another study, uridine-5-oxyacetic acid at the position 34 of tRNA Val displayed a similar Watson–Crick-like pair 27 with guanosine greatly displaced towards the minor groove of a codon–anticodon helix when compared with our structure ( Supplementary Fig. 4c left). On the basis of these 30S models, it was reasoned that modifications stabilized enol tautomers of uracil to keep the Watson–Crick-like geometry. It should also be mentioned here that although these structures gave important insights into how tRNA modifications can influence base-pairing interactions in the decoding centre, they described a partial system of decoding where roles of a full-length tRNA and the large ribosomal subunit could not be taken into consideration. At the same time, proper understanding of most often subtle fine-tuning effects of tRNA modifications on the codon–anticodon pairing geometries would require advanced experimental system consisting of full-length ligands and complete ribosome.

The novel pairing interaction at the third position of the codon–anticodon duplex described in this work deepens our understanding of principles embedded into translation of the genetic code on the ribosome. In agreement with the ‘modified wobble hypothesis’, our data show that the shape of the ‘wobble’ base pair is jointly defined by the ribosome environment and the tRNA modifications. In the observed case, such synergy gave rise to the novel base-pairing pattern, never observed before in the tRNA–mRNA duplexes. From the observation we can derive both a wider spatial tolerance of the wobble base-pair environment than expected before and, on the other hand, a certain degree of strictness imposed on the base pair, forcing it into the conformation unusual for a relaxed duplex.

Together with our preceding models describing G·U, U·G, A·C and A·A mismatches in codon–anticodon duplexes bound in the 70S decoding centre, the present models with pyrimidine–pyrimidine U·U mismatches consolidate the translation fidelity mechanism put forward by us earlier 29 , 30 . In this mechanism, the ribosome responds identically on binding of cognate or near-cognate tRNA by enveloping the codon–anticodon duplex in a rigid universal mould of the ‘closed’ decoding centre, which favours the Watson–Crick geometry of codon–anticodon base pairs. It is crucial to emphasize that when near-cognate tRNA with a mismatch to the mRNA codon binds to the decoding centre (during initial selection and proofreading steps), the number of hydrogen bonds between the minor groove of the near-cognate codon–anticodon helix and the ‘closed’ decoding centre elements is the same as would be the case during binding of the cognate tRNA. Therefore, the ribosome is incapable to distinguish between these states by the minor groove geometry of codon–anticodon base pairs. However, in contrast to cognate tRNAs that will stably pair to the mRNA codon by canonical Watson–Crick interactions, near-cognate tRNAs with a mismatch to the codon will be more likely to dissociate from the ribosome because of the strict restraints imposed by the tertiary structure of tRNA and elements of the ‘closed’ decoding centre on the codon–anticodon helix. This pressure constitutes the discriminatory force by preventing the following conformational changes: (a) widening of the codon–anticodon helix needed to accommodate bulky non-canonical pairs (for example, A·A pair); (b) narrowing of the codon–anticodon helix necessary for proper pairing interaction in pyrimidine–pyrimidine pairs (for example, U·U pair); or (c) shift of nucleobases towards minor or major groove, characteristic of, for example, wobble G·U pair. The third base pair of the codon–anticodon duplex contributes in a different manner, compared with the first two positions. Its role in decoding is linked to the base pair nature, the indirect restraints imposed by the decoding centre and the presence of the modification groups that influence conformations of the tRNA ASL and the codon–anticodon helix. An additional discriminatory role at the proofreading step can be performed by the tails of some ribosomal proteins that selectively stabilize cognate tRNA substrates 51 . The rare translational mistakes caused by the incorporation of near-cognate tRNAs are reasoned mostly by the ability of some tRNAs to form Watson–Crick-like base pairs via ionization or tautomerism 29 , or in some cases a mismatch randomly escapes discrimination by preserving geometry close to the Watson–Crick pair 30 . The present decoding mechanism further establishes that discrimination between tRNAs is primarily founded on spatial fit 29 , 30 , 48 rather than on the number of hydrogen bonds between the ‘closed’ decoding centre and the codon–anticodon duplex 35 .

Uncharged, native individual tRNA Lys SUU and tRNA fMet CAU from E. coli were purchased from Chemical Block (Russia). The mRNA constructs whose sequences are specified below were from Thermo Scientific (USA) and deprotected following the supplier procedure. All mRNA constructs contained identical sequence 5′-GGCAAGGAGGUAAAA-3′ at the 5′-end, which was followed by 5′-AUG AAA A 6 -3′ (mRNA-1), 5′-AUG AAG A 9 -3′ (mRNA-2), 5′-AUG UAA A 9 -3′ (mRNA-3) or 5′-AUG AUA A 9 -3′ (mRNA-4). Aminoglycoside antibiotic paromomycin was purchased from Sigma-Aldrich.

Purification of the ribosomes

Purification of the 70S ribosomes from strain HB8 of T. thermophilus was performed according to the protocol described in ref. 30 .

Complex formation

All ribosomal complexes were formed in 10 mM Tris-acetate pH 7.0, 40 mM KCl, 7.5 mM Mg(CH 3 COO) 2 , 0.5 mM dithiothreitol at 37 °C. For the cognate complexes ( Fig. 1c , complexes 1 and 2), the 70S ribosomes (3 μM) were pre-incubated with fivefold excess of mRNA-1 or mRNA-2 and threefold excess of tRNA fMet CAU for 15 min to fill the P-site. Then, tRNA Lys SUU was added at fivefold excess and incubation was continued for 30 min. Near-cognate complexes ( Fig. 1c , complexes 3 and 4) were prepared in a similar manner with the use of mRNA-3 and mRNA-4 constructs. Complexes with paromomycin were obtained by including the antibiotic (60 μM) into the incubation mixture containing 70S/tRNA fMet /mRNA-3/tRNA Lys SUU or 70S/tRNA fMet /mRNA-4/tRNA Lys SUU .

Crystallization and crystal treatment

Crystals were grown at 24 °C via vapour diffusion in sitting-drop plates (CrysChem, Hampton Research). The ribosomal complex (2 μl) containing 2.8 mM Deoxy Big Chaps (CalBioChem) was mixed with the equal volume of the crystallization solution (3.9–4.2% (w/v) PEG 20k, 3.9–4.2% (w/v) PEG550mme, 100 mM Tris-acetate pH 7.0, 100 mM KSCN). The crystals grew for 2–3 weeks and were then dehydrated by exchanging the reservoir for 60% (v/v) 2-methyl-2,4-pentanediol. Before freezing in the nitrogen stream, crystals were then cryoprotected by the addition of 30% (v/v) 2-methyl-2,4-pentanediol and 14 mM Mg(CH 3 COO) 2 .

Structure determination

Data for all complexes were collected at the PXI beamline of Swiss Light Source, Switzerland, at 100 K. A very low-dose mode was used and high redundancy data were collected 52 . The data were processed and scaled using XDS 53 . All crystals belong to space group P2 1 2 1 2 1 and contain two ribosomes per asymmetric unit. One of the previously published structures 29 , with tRNA, mRNA and metal ions removed, was used for refinement with Phenix 54 . The initial model was placed within each data set by rigid body refinement with each biopolymer chain as a rigid body. This was followed by initial coordinate refinement. The resulting electron density maps were inspected in Coot 55 , and the tRNA and mRNA ligands were built in. During several cycles of manual rebuilding followed by coordinate and isotropic B-factor refinement, magnesium ions were added and the final refinement round took place. The data collection and refinement statistics are presented in Table 1 .

Additional information

Accession codes: The atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession codes: 5E7K , 5E81 , 5EL4 , 5EL5 , 5EL6 and 5EL7 .

How to cite this article: Rozov, A. et al. Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code. Nat. Commun. 7:10457 doi: 10.1038/ncomms10457 (2016).

Accession codes

Protein data bank.

Machnicka, M. A. et al. MODOMICS: a database of RNA modification pathways--2013 update. Nucleic Acids Res. 41 , D262–D267 (2013).

Article   CAS   Google Scholar  

El Yacoubi, B., Bailly, M. & de Crecy-Lagard, V. Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Annu. Rev. Genet. 46 , 69–95 (2012).

Nishimura, S. & Watanabe, K. The discovery of modified nucleosides from the early days to the present: a personal perspective. J. Biosci. 31 , 465–475 (2006).

Agris, P. F., Vendeix, F. A. & Graham, W. D. tRNA's wobble decoding of the genome: 40 years of modification. J. Mol. Biol. 366 , 1–13 (2007).

Cantara, W. A. et al. The RNA modification database, RNAMDB: 2011 update. Nucleic Acids Res. 39 , D195–D201 (2011).

Durant, P. C., Bajji, A. C., Sundaram, M., Kumar, R. K. & Davis, D. R. Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: the effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A. Biochemistry 44 , 8078–8089 (2005).

Stuart, J. W., Koshlap, K. M., Guenther, R. & Agris, P. F. Naturally-occurring modification restricts the anticodon domain conformational space of tRNA(Phe). J. Mol. Biol. 334 , 901–918 (2003).

Crick, F. H. Codon—anticodon pairing: the wobble hypothesis. J. Mol. Biol. 19 , 548–555 (1966).

Yarian, C. et al. Accurate translation of the genetic code depends on tRNA modified nucleosides. J. Biol. Chem. 277 , 16391–16395 (2002).

Bjork, G. R. et al. Transfer RNA modification. Annu. Rev. Biochem. 56 , 263–287 (1987).

Agris, P. F. Wobble position modified nucleosides evolved to select transfer RNA codon recognition: a modified-wobble hypothesis. Biochimie 73 , 1345–1349 (1991).

Beuning, P. J. & Musier-Forsyth, K. Transfer RNA recognition by aminoacyl-tRNA synthetases. Biopolymers 52 , 1–28 (1999).

Grosjean, H. & Bjork, G. R. Enzymatic conversion of cytidine to lysidine in anticodon of bacterial isoleucyl-tRNA--an alternative way of RNA editing. Trends Biochem. Sci. 29 , 165–168 (2004).

Bjork, G. R. et al. Transfer RNA modification: influence on translational frameshifting and metabolism. FEBS Lett. 452 , 47–51 (1999).

Urbonavicius, J., Qian, Q., Durand, J. M., Hagervall, T. G. & Bjork, G. R. Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J. 20 , 4863–4873 (2001).

Thiaville, P. C. & de Crecy-Lagard, V. The emerging role of complex modifications of tRNA in signaling pathways. Microb. Cell 2 , 1–4 (2015).

Kellogg, D. A., Doctor, B. P., Loebel, J. E. & Nirenberg, M. W. RNA codons and protein synthesis. IX. Synonym codon recognition by multiple species of valine-, alanine-, and methionine-sRNA. Proc. Natl Acad. Sci. USA 55 , 912–919 (1966).

Article   CAS   ADS   Google Scholar  

Crick, F. H. The origin of the genetic code. J. Mol. Biol. 38 , 367–379 (1968).

Raba, M. et al. Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts. Eur. J. Biochem. 97 , 305–318 (1979).

Sprinzl, M. & Vassilenko, K. S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 33 , D139–D140 (2005).

Armengod, M. E. et al. Enzymology of tRNA modification in the bacterial MnmEG pathway. Biochimie 94 , 1510–1520 (2012).

Kumar, R. K. & Davis, D. R. Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability. Nucleic Acids Res. 25 , 1272–1280 (1997).

Ogle, J. M. et al. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292 , 897–902 (2001).

Murphy, F. V. T. & Ramakrishnan, V. Structure of a purine-purine wobble base pair in the decoding center of the ribosome. Nat. Struct. Mol. Biol. 11 , 1251–1252 (2004).

Murphy, F. V. t., Ramakrishnan, V., Malkiewicz, A. & Agris, P. F. The role of modifications in codon discrimination by tRNA(Lys)UUU. Nat. Struct. Mol. Biol. 11 , 1186–1191 (2004).

Vendeix, F. A. et al. Human tRNA(Lys3)(UUU) is pre-structured by natural modifications for cognate and wobble codon binding through keto-enol tautomerism. J. Mol. Biol. 416 , 467–485 (2012).

Weixlbaumer, A. et al. Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines. Nat. Struct. Mol. Biol. 14 , 498–502 (2007).

Jenner, L. B., Demeshkina, N., Yusupova, G. & Yusupov, M. Structural aspects of messenger RNA reading frame maintenance by the ribosome. Nat. Struct. Mol. Biol. 17 , 555–560 (2010).

Demeshkina, N., Jenner, L., Westhof, E., Yusupov, M. & Yusupova, G. A new understanding of the decoding principle on the ribosome. Nature 484 , 256–259 (2012).

Rozov, A., Demeshkina, N., Westhof, E., Yusupov, M. & Yusupova, G. Structural insights into the translational infidelity mechanism. Nat. Commun. 6 , 7251 (2015).

Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313 , 1935–1942 (2006).

Fernandez, I. S. et al. Unusual base pairing during the decoding of a stop codon by the ribosome. Nature 500 , 107–110 (2013).

Schmeing, T. M., Voorhees, R. M., Kelley, A. C. & Ramakrishnan, V. How mutations in tRNA distant from the anticodon affect the fidelity of decoding. Nat. Struct. Mol. Biol. 18 , 432–436 (2011).

Holley, R. W. et al. Structure of a ribonucleic acid. Science 147 , 1462–1465 (1965).

Ogle, J. M., Murphy, F. V., Tarry, M. J. & Ramakrishnan, V. Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111 , 721–732 (2002).

Yokoyama, S. et al. Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon. Proc. Natl Acad. Sci. USA 82 , 4905–4909 (1985).

Mazumdar, S. K. & Saenger, W. Molecular structure of poly-2-thiouridylic acid, a double helix with non-equivalent polynucleotide chains. J. Mol. Biol. 85 , 213–219 (1974).

Demeshkina, N., Jenner, L., Yusupova, G. & Yusupov, M. Interactions of the ribosome with mRNA and tRNA. Curr. Opin. Struct. Biol. 20 , 325–332 (2010).

Nakamura, Y. Codon Usage Database http://www.kazusa.or.jp/codon/ (2007).

Frédéric Dardel, F. K. Bioinformatics: Genomics and Post-Genomics John Wiley & Sons, Ltd. (2006).

Takai, K. & Yokoyama, S. Roles of 5-substituents of tRNA wobble uridines in the recognition of purine-ending codons. Nucleic Acids Res. 31 , 6383–6391 (2003).

Egli, M. & Sarkhel, S. Lone pair-aromatic interactions: to stabilize or not to stabilize. Acc. Chem. Res. 40 , 197–205 (2007).

Wohlgemuth, I., Pohl, C., Mittelstaet, J., Konevega, A. L. & Rodnina, M. V. Evolutionary optimization of speed and accuracy of decoding on the ribosome. Philos. Trans. R. Soc. Lond. B Biol. Sci. 366 , 2979–2986 (2011).

Kramer, E. B. & Farabaugh, P. J. The frequency of translational misreading errors in E. coli is largely determined by tRNA competition. RNA 13 , 87–96 (2007).

Johansson, M., Zhang, J. & Ehrenberg, M. Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection. Proc. Natl Acad. Sci. USA 109 , 131–136 (2012).

Manickam, N., Nag, N., Abbasi, A., Patel, K. & Farabaugh, P. J. Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors. RNA 20 , 9–15 (2014).

Moazed, D. & Noller, H. F. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16S rRNA. J. Mol. Biol. 211 , 135–145 (1990).

Demeshkina, N., Jenner, L., Westhof, E., Yusupov, M. & Yusupova, G. New structural insights into the decoding mechanism: translation infidelity via a G.U pair with Watson-Crick geometry. FEBS Lett. 587 , 1848–1857 (2013).

Westhof, E., Yusupov, M. & Yusupova, G. Recognition of Watson-Crick base pairs: constraints and limits due to geometric selection and tautomerism. F1000Prime Rep. 6 , 19 (2014).

Article   Google Scholar  

Grosjean, H., de Crecy-Lagard, V. & Marck, C. Deciphering synonymous codons in the three domains of life: co-evolution with specific tRNA modification enzymes. FEBS Lett. 584 , 252–264 (2010).

Jenner, L., Demeshkina, N., Yusupova, G. & Yusupov, M. Structural rearrangements of the ribosome at the tRNA proofreading step. Nat. Struct. Mol. Biol. 17 , 1072–1078 (2010).

Mueller, M., Wang, M. & Schulze-Briese, C. Optimal fine phi-slicing for single-photon-counting pixel detectors. Acta Crystallogr. D Biol. Crystallogr. 68 , 42–56 (2012).

Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66 , 125–132 (2010).

Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66 , 213–221 (2010).

Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66 , 486–501 (2010).

Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336 , 1030–1033 (2012).

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Acknowledgements

We thank the staff of the PXI beamline at the Swiss Light Source (Switzerland) for the advices in the synchrotron data collection. We are also thankful to Henry Grosjean for critical reading of the manuscript and constructive discussions. This work was supported by the French National Research Agency ANR-11-BSV8-006 01 (to G.Y.), the European Research Council advanced grant 294312 (to M.Y.), the Foundation for Medical Research in France grant FDT20140930867 (to I.K.) and the Russian Government Program of Competitive Growth of Kazan Federal University (to I.K., M.Y. and G.Y.). This work was also supported by the LABEX: ANR-10-LABX-0036_NETRNA and benefits from a funding from the state managed by the French National Research Agency as part of the Investments for the future program (to E.W.).

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Alexey Rozov and Natalia Demeshkina: These authors contributed equally to the work.

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Department of Integrated Structural Biology, Institute of Genetics and Molecular and Cellular Biology, INSERM, U964; CNRS/University of Strasbourg,

Alexey Rozov, Natalia Demeshkina, Iskander Khusainov, Marat Yusupov & Gulnara Yusupova

CNRS/University of Strasbourg, UMR7104, 1 rue Laurent Fries, BP 10142, Illkirch, 67404, France

Institute of Fundamental Medicine and Biology, Kazan Federal University, Karl Marx 18, Kazan, 420012, Russia

Iskander Khusainov

Architecture and Reactivity of RNA, Institute of Molecular and Cellular Biology of the CNRS, University of Strasbourg, UPR9002, 15 rue Rene Descartes, Strasbourg, 67084, France

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A.R. and N.D. conducted the experiments. I.K. purified ribosomes and assisted in X-ray data collection. A.R., N.D., E.W. and G.Y. interpreted the structures. A.R., N.D., I. K., E.W., M. Y. and G.Y. contributed to the final version of the paper.

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Correspondence to Gulnara Yusupova .

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Rozov, A., Demeshkina, N., Khusainov, I. et al. Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code. Nat Commun 7 , 10457 (2016). https://doi.org/10.1038/ncomms10457

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Biology Notes Online

The Wobble Hypothesis – Definition, Exaplanation, Importance

Table of Contents

What is Wobble Hypothesis?

The Wobble Hypothesis, proposed by Francis Crick in 1966, provides an explanation for the degeneracy of the genetic code. Degeneracy refers to the fact that multiple codons can code for the same amino acid. According to the Wobble Hypothesis, the precise pairing between the bases of the codon and the anticodon of tRNA occurs only for the first two bases of the codon. However, the pairing between the third base of the codon and the anticodon can exhibit some flexibility or “wobble.”

In other words, the third base of the codon and the anticodon can sway or move unsteadily, allowing for non-standard base pairing. This phenomenon enables a single tRNA molecule to recognize and bind to multiple codons, despite differences in their third base. As a result, although there are 61 codons that code for amino acids, the number of tRNA molecules is significantly lower (around 40) due to wobbling.

Wobble base pairs play a crucial role in RNA secondary structure and are critical for accurate translation of the genetic code. The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). These wobble base pairs exhibit thermodynamic stability comparable to that of Watson-Crick base pairs.

In the genetic code, there are 64 possible codons, out of which three are stop codons that terminate translation. If canonical Watson-Crick base pairing were required for each codon, it would necessitate 61 different types of tRNA molecules. However, most organisms have fewer than 45 types of tRNA. The Wobble Hypothesis explains how some tRNA molecules can recognize multiple synonymous codons, which encode the same amino acid.

Crick proposed that the 5′ base on the anticodon, which binds to the 3′ base on the mRNA codon, is more flexible spatially compared to the other two bases. This flexibility allows for non-standard base pairing and small conformational adjustments, resulting in the overall pairing geometry of tRNA anticodons.

Overall, the Wobble Hypothesis provides a mechanism to account for the degeneracy of the genetic code and the ability of a limited number of tRNA molecules to recognize and bind to multiple codons. The wobbling of the third base allows for greater flexibility in genetic coding while maintaining the accuracy of translation.

Definition of Wobble Hypothesis

The Wobble Hypothesis proposes that the third base of a codon and the anticodon of tRNA can exhibit flexibility or “wobble” in their base pairing, allowing a single tRNA molecule to recognize and bind to multiple codons, contributing to the degeneracy of the genetic code.

The Wobble Hypothesis

The Wobble Hypothesis proposes that the base at the 5′ end of the anticodon in tRNA is not as constrained spatially as the other two bases. This flexibility allows it to form hydrogen bonds with multiple bases located at the 3′ end of a codon in mRNA. The wobble hypothesis states that:

• The first two bases of the codon and the corresponding bases in the anticodon form normal Watson-Crick base pairs.
• The third position in the codon follows less strict base-pairing rules, leading to non-canonical pairing or “wobble.”
• The relaxed base-pairing requirement at the third position enables a single tRNA molecule to pair with more than one mRNA triplet.
• The specific rules for wobble pairing are: U in the first position of the anticodon can recognize A or G in the codon, G can recognize U or C, and I (inosine) can recognize U, C, or A.
• These characteristics led Francis Crick to propose the wobble hypothesis, which explains the flexible base-pairing interactions observed in the genetic code.

The coding specificity of the genetic code primarily depends on the first two bases of the codon, which form strong Watson-Crick base pairs with the anticodon of tRNA. The first nucleotide in the anticodon determines how many nucleotides the tRNA can distinguish when reading the codon in the 5′ to 3′ direction.

If the first nucleotide in the anticodon is C or A, the pairing is specific, and only one specific codon can be recognized by that tRNA. However, if the first nucleotide is U or G, the pairing is less specific, allowing interchangeability between two bases in the codon. In the case of inosine as the first nucleotide, it exhibits true wobble properties, enabling it to pair with any of three bases in the original codon.

Due to the specificity of the first two nucleotides in the codon, if an amino acid is coded for by multiple anticodons and those anticodons differ in the second or third position (first or second position in the codon), a different tRNA is required for each anticodon.

To satisfy all possible codons (61 excluding three stop codons), a minimum of 32 tRNA molecules is required. This includes 31 tRNAs for the amino acids and one for the initiation codon.

The Wobble Hypothesis provides a key understanding of the flexibility and degeneracy of the genetic code, allowing a limited number of tRNA molecules to recognize and bind to multiple codons while maintaining the accuracy of protein translation.

Wobble base pairs

Wobble base pairs are specific pairings between nucleotides in RNA molecules that deviate from the standard Watson-Crick base pair rules. They play a crucial role in RNA structure and translation. Here are some key points about wobble base pairs:

• Wobble base pairs involve non-standard pairings between nucleotides in specific positions.
• The four main wobble base pairs identified in RNA are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C).
• The use of “I” for hypoxanthine maintains consistency in nucleic acid nomenclature since hypoxanthine is the nucleobase of inosine.
• Among the wobble base pairs, inosine exhibits the most significant characteristics. If inosine is present as the first nucleotide in the anticodon of tRNA, it can pair with any of three bases (adenine, cytosine, or uracil) in the corresponding codon on mRNA.
• Inosine’s ability to wobble allows a single tRNA molecule with an inosine-containing anticodon to recognize multiple codons, expanding the flexibility of the genetic code.

Wobble base pairs introduce a level of versatility in RNA interactions, particularly during translation. They enable a reduced number of tRNA molecules to recognize multiple codons, compensating for the degeneracy of the genetic code. The wobble hypothesis, which explains these non-standard pairings, provides insights into the efficiency and accuracy of protein synthesis.

Short Exaplanation of The Wobble Hypothesis

Let’s dive into the Wobble Hypothesis step by step.

The Wobble Hypothesis is a concept that explains how the genetic code, stored in the form of nucleotides, is translated into proteins by the ribosome. To understand this hypothesis, we need to know a little bit about codons and anticodons.

Codons are sequences of three nucleotides in mRNA, and each codon corresponds to a specific amino acid or a stop signal. On the other hand, anticodons are sequences of three nucleotides in tRNA that bind to the codon during protein synthesis.

According to the Wobble Hypothesis, the base at the 5′ end of the anticodon is not as restricted in its pairing as the other two bases. This means that the first two bases of the codon and anticodon form normal hydrogen bond pairs, following the usual base-pairing rules (A with U, G with C). However, at the third position of the codon, the rules are more relaxed, and non-canonical pairing can occur.

In other words, the third base of the codon and the first base of the anticodon can form “wobble” pairs that do not strictly follow the A-U and G-C base-pairing rules. This flexibility allows the anticodon of a single tRNA molecule to recognize and bind to more than one codon with different nucleotide sequences at the third position.

To give you some examples of the wobble pairing rules:

• If the first base of the anticodon is U, it can recognize codons with A or G as the third base.
• If the first base of the anticodon is G, it can recognize codons with U or C as the third base.
• If the first base of the anticodon is I (Inosine), it can recognize codons with U, C, or A as the third base.

By allowing this “wobble” or relaxed base-pairing, the cell can minimize the number of tRNA molecules needed for protein synthesis. It provides flexibility and efficiency in translating the genetic code.

So, in summary, the Wobble Hypothesis proposes that the third base of the codon and the first base of the anticodon can form non-standard base pairs, leading to a more flexible set of base-pairing rules at the third position of the codon. This flexibility allows a single tRNA molecule to recognize and bind to multiple codons with different nucleotide sequences at the third position, optimizing protein synthesis.

Importance of the Wobble Hypothesis

The wobble hypothesis holds significant importance in understanding the efficiency and accuracy of protein synthesis. Here are key points highlighting the importance of the wobble hypothesis:

• Broad specificity with limited tRNAs: Our bodies possess a limited number of tRNA molecules. The wobble hypothesis allows a single tRNA to recognize multiple codons due to non-standard pairings at the wobble position. This broad specificity enables efficient translation with a smaller set of tRNAs.
• Facilitation of biological functions: Wobble base pairs have been extensively studied in organisms such as Escherichia coli ( E. coli ), demonstrating their role in various biological processes. They contribute to the accuracy of translation and protein synthesis.
• Comparable thermodynamic stability: Wobble base pairs exhibit thermodynamic stability similar to Watson-Crick base pairs. This stability ensures the integrity of RNA secondary structures and promotes reliable translation of the genetic code.
• Essential for RNA secondary structure: Wobble base pairs play a fundamental role in the formation of RNA secondary structures. They contribute to the stability and folding of RNA molecules, enabling the proper functioning of RNA in various cellular processes.
• Faster dissociation and protein synthesis: The wobble base pairing allows faster dissociation of tRNA from mRNA during the translation process. This rapid dissociation promotes efficient protein synthesis by facilitating the movement of ribosomes along the mRNA template.
• Minimizing errors in genetic code interpretation: The existence of wobble minimizes the impact of certain errors in the genetic code. If a codon is misread during transcription, wobble allows the tRNA to still recognize and correctly translate the codon, maintaining the appropriate amino acid sequence during protein synthesis. This reduces the potential damage that can arise from occasional errors in the reading of the genetic code.

Quiz on Wobble Hypothesis

What does the Wobble Hypothesis explain? a) The structure of DNA b) The replication of DNA c) The flexibility in the pairing of the third base of the codon d) The synthesis of proteins

Who proposed the Wobble Hypothesis? a) Rosalind Franklin b) James Watson c) Francis Crick d) Maurice Wilkins

According to the Wobble Hypothesis, which position of the codon shows flexibility in base pairing? a) First b) Second c) Third d) Fourth

Which base can pair with multiple bases according to the Wobble Hypothesis? a) Adenine b) Cytosine c) Guanine d) Thymine

The Wobble Hypothesis helps to explain why: a) There are more codons than amino acids b) There are more amino acids than codons c) Codons are always of fixed length d) DNA is double-stranded

Which of the following is NOT a valid wobble pairing? a) G-U b) A-U c) I-A d) C-G

The Wobble Hypothesis reduces the need for: a) Multiple DNA strands b) Multiple types of amino acids c) Multiple types of tRNAs d) Multiple types of ribosomes

Inosine (I) can pair with which of the following bases? a) Adenine b) Cytosine c) Both Adenine and Cytosine d) Neither Adenine nor Cytosine

The Wobble Hypothesis is primarily associated with: a) DNA replication b) Transcription c) Translation d) DNA repair

The flexibility in base pairing, as proposed by the Wobble Hypothesis, occurs between: a) mRNA codon and DNA template b) mRNA codon and tRNA anticodon c) tRNA anticodon and DNA template d) tRNA anticodon and ribosomal RNA

What is the wobble hypothesis?

The wobble hypothesis proposes that the base at the 5′ end of the anticodon in tRNA is not as strictly paired with the corresponding base in the mRNA codon, allowing for non-standard or wobble base pairings.

Why is it called the “wobble” hypothesis?

It is named the “wobble” hypothesis because the base at the wobble position is not spatially confined like the other two bases in the anticodon, allowing it to wobble or move unsteadily and form non-standard base pairs.

How does the wobble hypothesis explain degeneracy in the genetic code?

The wobble hypothesis suggests that the relaxed base-pairing rules at the third position of the codon allow a single tRNA molecule to recognize more than one codon. This accounts for the degeneracy or redundancy of the genetic code.

What are the main wobble base pairs?

The main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C).

Why is hypoxanthine used in wobble base pairs?

Hypoxanthine is used to represent wobble base pairs because it is the nucleobase of inosine, which displays the true qualities of wobble by allowing for pairing with multiple bases in the original codon.

How does the wobble hypothesis impact protein synthesis?

The wobble base pairing allows for faster dissociation of tRNA from mRNA during protein synthesis, facilitating the movement of ribosomes and enhancing the efficiency of translation.

What role do wobble base pairs play in RNA secondary structure?

Wobble base pairs are fundamental in RNA secondary structure. They contribute to the stability and folding of RNA molecules, influencing their overall structure and function.

Does the wobble hypothesis affect the accuracy of the genetic code?

Yes, the wobble hypothesis helps to minimize errors in the interpretation of the genetic code. It ensures that even if there is a mismatch at the wobble position, the correct amino acid can still be incorporated during protein synthesis.

How does the wobble hypothesis impact the number of tRNA molecules needed?

The wobble hypothesis allows a single tRNA molecule to recognize multiple codons due to non-standard pairings. This broad specificity reduces the number of unique tRNA molecules required for translation.

How has the wobble hypothesis contributed to our understanding of molecular biology?

The wobble hypothesis has provided insights into the efficiency and accuracy of protein synthesis, RNA structure, and the functioning of the genetic code. It has enhanced our understanding of how cells optimize resources and maintain fidelity in the complex process of translating genetic information into functional proteins.

• https://microbenotes.com/the-wobble-hypothesis/
• https://en.wikipedia.org/wiki/Wobble_base_pair
• https://teaching.ncl.ac.uk/bms/wiki/index.php/Wobble_Hypothesis
• https://link.springer.com/referenceworkentry/10.1007%2F978-3-642-11274-4_1692
• https://www.biologydiscussion.com/genetics/wobble-hypothesis-with-diagram-genetics/65163

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Wobble Pair

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• Henderson James Cleaves 11 , 12 , 13  

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A wobble pair, or wobble-base pair, is a hydrogen-bonded pairing between two nucleotides generally occurring between two RNA molecules. These base pairs are geometrically distinct from the canonical Watson–Crick-type base-pairing. The main wobble base pairs are hypoxanthine-uracil (I-U, where I represents inosine, the nucleoside formed from hypoxanthine), guanine-uracil (G-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). Wobble base pairs are of comparable thermodynamic stability to Watson–Crick base pairs. Wobble base pairs occur frequently in RNA secondary structure and are important for proper translation of the genetic code.

In 1966, Francis Crick proposed the Wobble hypothesis to account for the fact that most organisms do not seem to have as many tRNA molecules as would be required for complete translation of the genetic code. There are 64 possible codons in the genetic code. During translation, each of these codons requires a tRNA molecule with a...

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Earth-Life Science Institute (ELSI), Tokyo Institute of Technology, Tokyo, Japan

Henderson James Cleaves

Blue Marble Space Institute of Science, Washington, DC, USA

Center for Chemical Evolution, Georgia Institute of Technology, Atlanta, GA, USA

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Laboratoire d'Astrophysique de Bordeaux, University of Bordeaux, Pessac CX, France

Muriel Gargaud

Department of Astronomy, University of Massachusetts, Amherst, MA, USA

William M. Irvine

Centro Biología Molecular Severo Ochoa, Univ. Autónoma de Madrid Cantoblanco, Madrid, Spain

Ricardo Amils

Analytical-, Environm., Geo-Chemistry, Vrije Universiteit Brussel, VUB, Brussels, Belgium

Philippe Claeys

Earth-Life Science Institute, Tokyo Institute of Technology, WASHINGTON, DC, USA

Radio Astronomy, Paris Observatory, Paris, France

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Observatoire de Paris-Meudon, Meudon, France

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International Space Science Institute, Bern, Bern, Switzerland

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Michel Viso

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Cleaves, H.J. (2022). Wobble Pair. In: Gargaud, M., et al. Encyclopedia of Astrobiology. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-27833-4_5248-2

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The wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons

S. joakim näsvall.

Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden

Glenn R. Björk

According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo 5 U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo 5 U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo 5 U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo 5 U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo 5 U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo 5 U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.

INTRODUCTION

The genetic message is read by tRNAs that decode one triplet at a time. Of the 64 codons, 61 are sense codons and represent an amino acid in the final protein. Triplets with the same first two nucleosides constitute a codon box, and if all four codons represent one amino acid, such a box is called a family codon box. In all organisms there are eight family codon boxes ( Fig. 1 , shaded), and in Salmonella enterica serovar Typhimurium, six of them are decoded by tRNAs of which one has uridine-5-oxyacetic acid (cmo 5 U34) or its methylester (mcmo 5 U34) in position 34 (the wobble position) ( Fig. 2 ). These six family codon boxes are specific for leucine, valine, serine, proline, threonine, and alanine ( Fig. 1 , light shade). To read the four codons in such a family codon box, there are, besides the cmo 5 U34-containing tRNA, one (valine and alanine) or two (leucine, serine, threonine, and proline) additional isoacceptor tRNAs. One of these isoacceptors has G as the wobble nucleoside, and in four boxes (leucine, proline, threonine, and serine) the third isoacceptor has C as the wobble nucleoside ( Fig. 1 ). According to the wobble hypothesis ( Crick 1966 ), G34 base-pairs with C and U as the third nucleoside of the codon [denoted C(III) and U(III)], whereas C34 only base-pairs with G(III). Uridine as the wobble nucleoside cannot interact with a pyrimidine in the mRNA, since two pyrimidines are too “short” to form a base pair. Therefore, it was thought that the G34-containing tRNAs are essential for decoding the U- and C-ending codons. However, U as the wobble nucleoside is almost always modified, and the cmo 5 -modification and the related modification 5-methoxyuridine, mo 5 U, present in tRNA of Bacillus subtilis , is predicted to extend the wobble capacity to read not only A(III) and G(III), as predicted by the wobble hypothesis, but also U(III), but not C(III) ( Yokoyama et al. 1985 ). Thus, the G34-containing tRNAs seems to be required to decode the C-ending codons in these family codon boxes. Most in vitro experiments with Escherichia coli tRNAs or anticodon stem–loops (ASLs) support the theoretical considerations that a U reads A(III) and G(III) and that cmo 5 U enhances the wobble to include U(III), but not C(III) ( Oda et al. 1969 ; Ishikura et al. 1971 ; Mitra et al. 1979 ; Samuelsson et al. 1980 ; Takai et al. 1999 ; Phelps et al. 2004 ; Sørensen et al. 2005 ).

The genetic code. The eight codon boxes with shaded background are the family codon boxes, containing four codons encoding one amino acid (fourfold degenerate). The six lighter-shaded boxes contain tRNAs having cmo 5 U as wobble nucleoside. The boxes with white background are the mixed codon boxes. A circle corresponds to a codon read by a tRNA, and a line connecting two or more circles indicates that the same tRNA is able to read those codons. Filled circles indicate codon reading as predicted by the wobble hypothesis ( Crick 1966 ) or the revised wobble rules ( Yokoyama et al. 1985 ). Open circles indicate that those tRNAs are able to read also the C-ending codons (results presented in this study and in Näsvall et al. 2004 ). Next to the symbol for each tRNA is indicated which wobble nucleoside it contains. The letters within parentheses below the wobble nucleoside in the family boxes for proline, threonine, alanine, and valine indicate the last letter in the name of the genes encoding the corresponding tRNAs (e.g., tRNA Val cmo5UAC is encoded by the four genes val T , val U , val X , and val Y , and tRNA Pro GGG is encoded by the gene pro L ).

The proposed biosynthetic pathway for the synthesis of cmo 5 U and mcmo 5 U. (Gray arrows) Indicate the link between chorismic acid (or an unknown derivative of it) and different steps in the synthesis of cmo 5 U according to Näsvall et al. (2004) . (U) Uridine; (ho 5 U) 5-hydroxyuridine; (mo 5 U) 5-methoxyuridine; (cmo 5 U) uridine-5-oxyacetic acid; (mcmo 5 U) uridine-5-oxyacetic acid methyl ester. (Adapted from Näsvall et al. 2004 and reprinted with permission from the RNA Society ©2004.)

In contrast to the above-mentioned results obtained in vitro, there is evidence from in vivo experiments that cmo 5 U34-containing tRNAs base-pair also with C(III). A strain that lacks the G34-containing tRNA Pro GGG (the subscript indicates the sequence of the anticodon in the 5′→3′ direction) and the C34-containing tRNA Pro CGG and thus only has the cmo 5 U34-containing tRNA Pro cmo5UGG is viable, demonstrating that tRNA Pro cmo5UGG with cmo 5 U34 as the wobble nucleoside can read all four proline codons ( Näsvall et al. 2004 ). Based on a synergistic growth defect in mutants that lack tRNA Pro GGG and are hypo-modified in the wobble position of tRNA Pro cmo5UGG , the presence of cmo 5 U34 was suggested to promote an efficient reading of C- and U-ending proline codons ( Näsvall et al. 2004 ). Similarly, a strain having only tRNA Ala cmo5UGC with cmo 5 U34 as the wobble nucleoside is also viable ( Gabriel et al. 1996 ). It was recently shown that the binding of tRNA Ala cmo5UGC to GCC codons is only slightly weaker than binding to GCA, and that the kinetics of A-site binding at GCC is within the range for cognate interactions ( Kothe and Rodnina 2007 ). Thus, at least tRNA Pro cmo5UGG and tRNA Ala cmo5UGC are able to read codons ending with C(III), contrary to the theory and to most results obtained in vitro. However, the impact of cmo 5 U34 on decoding by tRNA Ala cmo5UGC was not addressed by Gabriel et al. (1996) or Kothe and Rodnina (2007) , since their analysis was performed with fully modified tRNA Ala cmo5UGC ( Gabriel et al. 1996 ; Kothe and Rodnina 2007 ). Here, we extend these studies to elucidate whether the cmo 5 U34-containing tRNAs specific for valine and threonine are also able to read the four codons in the corresponding family boxes and if the presence of cmo 5 U34 is required for such a reading in the family codon boxes specific for valine, alanine, and threonine.

To study the function of cmo 5 U34 in vivo, we need a way to manipulate the presence of cmo 5 U34 in tRNA. We have recently identified two genes ( cmoA and B ) whose products are required for the synthesis of cmo 5 U34 ( Näsvall et al. 2004 ). Deletion of the cmoB gene results in a complete absence of cmo 5 U34 in tRNA, and all of the cmo 5 U found in the wild type is present as the biosynthetic intermediate 5-hydroxyuridine (ho 5 U) ( Fig. 2 ; Näsvall et al. 2004 ). Therefore, we have changed the allelic state of the cmoB gene in our attempt to demonstrate the coding capacities of cmo 5 U versus ho 5 U in tRNAs specific for proline, alanine, and valine. Surprisingly, considering the wobble hypothesis and other predictions ( Crick 1966 ; Yokoyama et al. 1985 ), our results show that cmo 5 U is required for efficient decoding of G-ending codons by tRNA Ala cmo5UGC , tRNA Val cmo5UAC , and tRNA Pro cmo5UGG . Thus, whereas G as the wobble nucleoside can base-pair with U in mRNA, apparently the reverse pairing [U34-G(III)] requires a modification of uridine.

tRNA Thr cmo5UGU containing cmo 5 U34 cannot read all four threonine codons

S. enterica has three threonine isoacceptors (tRNA Thr cmo5UGU , tRNA Thr GGU , and tRNA Thr CGU ) (see Fig. 1 ). The G34-containing tRNA Thr GGU is encoded by the genes thrT and thrV and the C34-containing tRNA Thr CGU is encoded by the gene thrW ( Fig. 3A ). Strains lacking the genes encoding tRNA Thr GGU and tRNA Thr CGU were constructed by inserting a kanamycin resistance cassette flanked by FLP recombinase target sequences (FRTs) ( Datsenko and Wanner 2000 ) into the thrT , thrV , or thrW genes. The three resulting single mutants lacking either the C34-containing tRNA Thr CGU or one of the two genes encoding the G34-containing tRNA Thr GGU were all viable, with no apparent growth phenotype on solid rich medium at 37°C (data not shown).

Locations of tRNA genes in the S. enterica genome. ( A ) Threonine tRNAs. ( Upper line) The thrW gene, encoding tRNA Thr CGU . ( Middle line) The rrnD rRNA operon containing one of the two genes encoding tRNA Thr GGU . ( Lower line) The tufB operon, containing the gene encoding tRNA Thr cmo5UGC as well as the second gene encoding tRNA Thr GGU . ( B ) Valine tRNA genes. ( Upper and middle lines) The two tRNA operons containing the four genes encoding tRNA Val cmo5UAC . ( Lower line) The dicistronic valV , valW operon containing the two genes encoding tRNA Val GAC . ( C ) Alanine tRNAs. ( Upper line) The three rRNA operons rrnH , rrnA , and rrnB , containing the genes encoding tRNA Ala cmo5UGC , have the same basic organization except an additional tRNA gene ( aspU ) at the end of rrnH . ( Lower line) The alaW , alaX tRNA operon encoding tRNA Ala GGC . ( D ) Proline isoacceptors. ( Upper line) The monocistronic proL gene, encoding tRNA Pro GGG . ( Middle line) The proK gene, encoding tRNA Pro CGG . ( Lower line) The operon containing the gene encoding tRNA Pro cmo5UGG as well as three other tRNA genes. (Black arrows) tRNA genes encoding threonine, valine, alanine, or proline tRNAs; (dark gray arrows) other tRNA genes; (light gray arrows) other genes. The anticodons of the relevant tRNAs are written below the genes. The drawings are not to scale. The asterisk (*) after gltT (which encodes tRNA Glu mnm5s2UUC ) in A , middle line, indicates that the gene (STM3397) is not named in Salmonella . gltT is the name of an identical gene in the E. coli rrnB operon, which in Salmonella instead contains the genes ileU and alaU (asterisks in C ).

To test if tRNA Thr cmo5UGU is able to read all four threonine codons, we attempted to generate a mutant having tRNA Thr cmo5UGU as the only remaining threonine isoacceptor by deleting the two genes encoding the G34-containing tRNA Thr GGU ( thrT and V ) and the C34-containing tRNA Thr CGU (thrW ). Whereas construction of mutants with one remaining gene encoding the G34-containing tRNA Thr GGU was possible, combining mutations in both genes encoding this tRNA failed ( Table 1 ). These results suggest that a double mutant ( thrT thrV ) having only the cmo 5 U34- and C34-containing threonine isoacceptors is not viable and consequently indicates that tRNA Thr GGU is essential. Still, a few transductants appeared in the attempts to make the thrT thrV double mutant. Since it is known that in a growing culture of Salmonella , different loci can be transiently duplicated, the rare Km R transductants may have both the wild-type allele and the mutated allele of thrT or thrV . Indeed, all of the 37 tested transductants possessed both the wild-type and the thrT <> kan alleles. Moreover, purification of 10 different Km R transductants on nonselective plates revealed segregation of Km R and Km S clones as expected if the original transductant contains a duplication of the wild-type and the thrT <> kan alleles. We conclude that the G34-containing tRNA Thr GGU is essential, most likely because the cmo 5 U34-containing tRNA Thr cmo5UGU cannot recognize C-ending threonine codons.

The cmo 5 U-containing tRNA Thr cmo5UGU is unable to decode all four threonine codons

The cmo 5 U-containing tRNA Val cmo5UAC by itself can only support growth at an extremely reduced rate

The two genes valV and valW , encoding two slightly different tRNA Val GAC s, are present as a tandem repeat in a dicistronic operon with no other genes ( Fig. 3B ). A mutant (Δ valVW ) lacking tRNA Val GAC , and thus having only the cmo 5 U-containing tRNA Val cmo5UAC to read the four valine codons ( Fig. 1 ), was viable but showed a 70% reduction in growth rate ( Table 2 ). These results indicate that, similarly to tRNA Pro cmo5UGG ( Näsvall et al. 2004 ), tRNA Val cmo5UAC is also able to read all four valine codons, albeit with low efficiency.

tRNA Val cmo5UAC by itself supports growth at an extremely reduced rate

As earlier reported, cmoB mutants have ho 5 U34 instead of cmo 5 U34 in their tRNA ( Näsvall et al. 2004 ). To test the impact of such hypo-modification on the decoding capacity of tRNA Val cmo5UAC , we disrupted the cmoB gene in a strain lacking the G34-containing tRNA Val GAC . This strain is viable but showed a decrease in growth rate compared to the parent strain ( Table 2 ), and it also accumulated faster-growing suppressor mutants (data not shown). In addition to the slow-growth phenotype, cultures sometimes formed visible aggregates, which were caused by part of the population of cells forming long filaments (data not shown). Clearly, the presence of the cmo 5 -modification improves the decoding efficiency of tRNA Val cmo5UAC .

To test if increased levels of tRNA Val cmo5UAC would help mutants lacking tRNA Val GAC and cmo 5 U, we compared the growth of strains harboring either plasmid p815 ( O'Connor 2002 ) (carrying the E. coli valU operon, containing three genes encoding tRNA Val cmo5UAC and one gene encoding tRNA Lys mnm5s2UUU ) or plasmid pLG339 (vector control). Overexpression of tRNA Val cmo5UAC partially suppressed the growth phenotypes of the strains having only this tRNA ( Fig. 4 ). Also, overexpression of the hypo-modified tRNA Val ho5UAC improved the growth of the Δ valVW cmoB2 mutant, demonstrating that tRNA Val cmo5UAC at normal concentration is, indeed, dependent on the cmo 5 -modification.

Overexpression of tRNA Val cmo5UAC restores growth of a Δ valVW mutant. ( A ) Growth after 25 h of incubation at 37°C. (Sectors 1 – 4 ) Strains carrying pLG339 (vector control); (sectors 5 – 8 ) strains carrying p815 ( valU valX valY lysV ). The chromosomal genotypes are ( 1 , 8 ) LT2 (wt); ( 2 , 7 ) cmoB2<>cat ; ( 3 , 6 ) ΔvalVW ; ( 4 , 5 ) ΔvalVW cmoB2<>cat . ( B ) Sectors 3 and 4 of the same plate as in A , but after 44 h at 37°C. No suppressor mutants were apparent in this particular experiment. The relative colony sizes after 15 h of growth were (LT2/pLG339 and cmoB2 /pLG339) 1.0 ± 0.03; (LT2/p815) 1.0 ± 0.07; ( cmoB2 /p815) 1.0 ± 0.03; (Δ valVW /p815) 0.68 ± 0.04. Colonies of Δ valVW cmoB2 /p815 were visible but still too small to measure, and no colonies were visible of Δ valVW /pLG339 or Δ valVW cmoB2 /pLG339. After 25 h, colonies of ΔvalVW cmoB2 /p815 were ∼30% smaller than Δ valVW /p815, and after 44 h, ( B ) colonies of Δ valVW cmoB2 /pLG339 were approximately half the size compared to Δ valVW /pLG339.

In order to further study the efficiency of tRNA Val cmo5UAC in reading the four valine codons and the effect of having ho 5 U in place of cmo 5 U, we used the system described by Curran and Yarus (1989) to measure the in vivo A-site selection rates ( Fig. 5 ). The mutant lacking both tRNA Val GAC and cmo 5 U (Δ valVW cmoB2 <> frt ) was not included because of difficulties in keeping the culture suppressor free, but also because of the filamentous growth phenotype, which would produce unreliable OD values. As expected, the rate of A-site selection is severely reduced at all four codons in the strain lacking the G34-containing tRNA Val GAC ( Fig. 5 ). Most severely affected was the rate at the GUC codon. This is not surprising considering the fact that this strain lacks the tRNA (tRNA Val GAC ) that is the major tRNA recognizing GUC codons. The data show the relative efficiency of fully modified tRNA Val cmo5UAC at recognizing the different codons; it recognized GUA, GUU, and GUG with equal efficiency, while, as expected, it was quite poor at recognizing the GUC codon. Whereas the cmoB2 mutation did not influence the rate of valyl-tRNA Val selection to the GUU- and GUA-programmed ribosomal A-site ( Fig. 5 ), significant decreases in the rates at GUC and, unexpectedly, also at GUG codons were observed.

A-site selection rates at valine (GUN) codons. (*) Values in the cmoB2 mutant are significantly different from the control (LT2), as determined by a student's t -test (two sample, equal variance, p < 0.05). All values for the Δ valVW mutant are significantly different from LT2 ( p < 0.005). The values are averages of four experiments, with at least two independent cultures of each strain.

tRNA Ala cmo5UGC requires cmo 5 U34 for efficient wobble reading of GCG

The two identical genes alaX and alaW (sometimes referred to as alaW α and alaW β ) ( Fig. 3C ) encoding tRNA Ala GGC , are arranged as a tandem repeat in a single operon containing no other genes. A strain lacking tRNA Ala GGC (Δ alaXW ) is viable, as is also the case in E. coli ( Gabriel et al. 1996 ), but has a clear reduction in growth rate compared to the wild-type strain (LT2) ( Table 3 ), a phenotype that is further enhanced at higher temperature, seen as a decreased colony size on plates at 44°C (data not shown). These results indicate that, similarly to tRNA Pro cmo5UGG ( Näsvall et al. 2004 ), tRNA Ala cmo5UGC is able to read all four alanine codons, although not efficiently enough to support a maximum growth rate.

tRNA Ala cmo5UGC alone can support growth only at a reduced rate

When cmoB <> kan was transduced into a strain (Δ alaXW ) lacking tRNA Ala GGC , tiny colonies (barely visible without magnification) started to appear after 2 d of incubation at 37°C. A few larger colonies (∼0.2% of totally 409 transductants in one transduction) were also apparent, indicating the presence of suppressor mutants in some colonies. The “tiny” colonies were purified on selective medium and found to be viable but to accumulate suppressor mutations that partially restored growth ( Fig. 6 ). When we transduced a wild-type strain (LT2) with the same amount of the same phage lysate, we received about the same number of transductants as when strain GT7365 (Δ alaXW ) was used as recipient, but the obtained transductants showed a normal growth phenotype. These results show that a mutant lacking tRNA Ala GGC is viable even when it is hypo-modified at the wobble position in the only remaining alanine tRNA, but it has an extremely reduced growth rate, and mutations that partially restore growth are relatively frequent. If the growth phenotype would be caused by poor reading of one or more of the alanine codons, expression of more of the hypo-modified tRNA Ala cmo5UGC would allow the mutant to grow faster. To test this, we introduced the cmoB2 <> kan allele into strains harboring plasmid p70 ( Vila-Sanjurjo et al. 1999 ), carrying E. coli genes encoding tRNA Ala cmo5UGC and four other tRNAs (tRNA Asp QUC , tRNA Trp CCA , tRNA Ile GAU , and tRNA Thr GGU ) expressed from the tac promoter. The growth phenotypes (seen as relative colony sizes on plates) of these strains were compared to the corresponding plasmid-free strains ( Fig. 6 ). The relatively mild growth defect of the Δ alaXW strain, which has only the cmo 5 U34-containing alanine tRNA, seems to be fully suppressed, and the Δ alaXW cmoB2 <> kan mutant is partially suppressed by overexpression of tRNA Ala cmo5UGC . Thus tRNA Ala cmo5UGC , when expressed at normal levels, is very dependent on the presence of the modification for its ability to read some of the four codons, but less so if it is overexpressed. We also measured the A-site selection rate at the four alanine codons. The Δ alaXW cmoB2 mutant was considered too slow growing and unstable to be included in such an analysis. The cmoB2 mutant shows a large reduction in the rate of reading the GCG codon ( Fig. 7 ). The Δ alaXW mutant has a reduction in the rate of alanyl-tRNA entry on all four codons, and the most severe reduction is on the GCC codon. Taken together, these results show that fully modified tRNA Ala cmo5UGC reads GCA, GCG, and GCU efficiently and GCC poorly and that cmo 5 U improves reading of the G-ending codon.

Overexpression of hypo-modified tRNA Ala cmo5UGC from plasmid p70 can partially restore growth of a Δ alaXW cmoB2 mutant. ( A ) Growth of (sector 1 ) LT2/p70 (wild-type); (sector 2 ) cmoB2<>kan /p70; (sector 3 ) Δ alaXW /p70; and (sector 4 ) Δ alaXW cmoB2<>kan /p70 after 27 h at 37°C on an LA + tetracycline plate. The relative colony diameters (after 16 h) were (LT2/p70) 1.0 ± 0.05; ( cmoB2 /p70) 0.96 ± 0.04; and (Δ alaXW /p70) 0.92 ± 0.07. The colonies of Δ alaXW cmoB2 /p70 were visible but too small to measure. ( B ) Same as in A , but the strains do not contain any plasmid, and the plate is LA without any antibiotic. The relative colony diameters (after 16 h) were (LT2) 1.0 ± 0.07; ( cmoB2 ) 1.0 ± 0.01; and (Δ alaXW ) 0.71 ± 0.04. At the time of the size measurements, no single colonies of the Δ alaXW cmoB2 mutant had appeared, but after 27 h, very tiny colonies (<0.1 mm in diameter) as well as some faster-growing colonies (still too small to be clearly visible in the picture) could be seen. ( C ) Sector 4 of the plate in B , but after 75 h. The absolute majority of the colonies of the Δ alaXW cmoB2 mutant were still <0.4 mm in diameter, while a few larger colonies ranging in sizes between ∼0.5 and 2 mm were visible.

A-site selection rates at alanine (GCN) codons. The asterisks (*) indicate values from the cmoB2 mutant that are different from the control (LT2), as determined by a student's t -test [two sample, equal variance; (*) p < 0.05, (***) p < 0.0005]. All values from the Δ alaXW mutant are significantly different from LT2 ( p < 0.0005). The values are averages from four experiments.

cmo 5 U34 in tRNA Pro cmo5UGG mainly enhances wobble reading of G

A mutant having only the cmo 5 U-contatining tRNA Pro cmo5UGG is viable without any apparent phenotype, demonstrating that this tRNA reads efficiently all four proline codons. If this tRNA in such a mutant contains ho 5 U instead of cmo 5 U34, a clear reduction in growth rate caused by the hypo-modification is observed ( Näsvall et al. 2004 ). Furthermore, a mutant lacking the G34-containing tRNA Pro GGG and thus having the cmo 5 U34- and C34-containing tRNAs has a significant growth rate reduction in conjunction with hypo-modification of the wobble nucleoside in tRNA Pro cmo5UGG ( Näsvall et al. 2004 ). Based on these data and the theoretical prediction that cmo 5 U34 reads U-ending codons (but not C-ending codons) ( Yokoyama et al. 1985 ), we suggested that the reason for the observed phenotypes of the various mutants being deficient in tRNA Pro and cmo 5 U was the slower reading of mainly the U- and C- ending proline codons ( Näsvall et al. 2004 ). To verify this suggestion, we measured the A-site selection rates on each of the four proline codons. Lack of tRNA Pro CGG and tRNA Pro GGG leads to a large reduction in the rate of reading all four proline codons ( Fig. 8 , cf. LT2 and Δ proKL ). This is not surprising, since the two missing tRNAs together make up about two-thirds of the total proline tRNA pool ( Dong et al. 1996 ) and should normally read most of the CCC, CCU, and CCG codons. Similarly to the alanine tRNA ( Fig. 7 ), the largest effect of hypo-modification of tRNA Pro cmo5UGG ( Fig. 8 , cf. LT2 and cmoB2 and Δ proKL and cmoB2 Δ proKL ) seems to be a reduced rate of reading CCG. We also measured the effect of not having cmo 5 U on the A-site selection rates in mutants only lacking the C34-containing tRNA Pro CGG ( proK <> frt ). In two separate experiments, the rate of reading the CCG codon in the cmoB2 mutant was reduced by 42% and 39%, respectively (data not shown), further strengthening our observations that cmo 5 U34 is important for recognizing the G-ending proline codon.

A-site selection rates at proline (CCN) codons. The asterisks (*) indicate values from the cmoB2 mutant that are different from the control (LT2), as determined by a student's t -test [two sample, equal variance; (*) p < 0.05]. All values for the Δ proL proK <> frt and Δ proL proK <> frt cmoB2 <> frt mutants are significantly different from LT2 ( p < 0.01). The values are averages from at least three experiments. For simplicity, Δ proL proK <> frt is written Δ proKL .

In this study, we show that the function of the modified nucleoside cmo 5 U34 is different from what has previously been hypothesized and that the impact on hypo-modification of the wobble position is different in different cmo 5 U34-containing tRNAs. According to a theoretical model, cmo 5 U is predicted to allow reading of U-ending codons ( Yokoyama et al. 1985 ). This model was based on how the modification (cmo 5 or mo 5 ), by interacting with the 5′-phosphate, affects the equilibrium between two different conformations (C2′-endo and C3′-endo) of the ribose moiety of synthetic nucleotides in solution. 5-Hydroxy uridine would not be able to make this interaction and would thus have decoding properties similar to uridine, which should only read A- and G-ending codons according to the wobble hypothesis ( Crick 1966 ). The model did not explain why some cmo 5 U-containing tRNAs can read C-ending codons, and, in fact, it was predicted that a cmo 5 U-C pair would be impossible due to steric repulsion between ribose 34 and ribose 35 ( Yokoyama et al. 1985 ). Moreover, this model would predict that a tRNA having ho 5 U in place of cmo 5 U would have a dramatically reduced rate of reading U-ending (and probably also C-ending in the cases where it does happen) codons, while the rates of reading the A- and G-ending codons should not be too much affected. However, our results do not support such a hypothesis, since the largest effect of hypo-modification of three tested tRNAs is on the rate of reading G-ending codons, while the effects (if any) on C- and U-ending codons are minor. This leads us to question the validity of the above model and suggests an alternative molecular mechanism for the decoding by cmo 5 U.

Interestingly, the effects on the growth rates or viability when removing all other tRNA isoacceptors for the different amino acids are quite different. One extreme is the cmo 5 U-containing threonine tRNA, which cannot at all support growth of a mutant lacking the G34-containing threonine isoacceptor ( Table 1 ). This is similar to previously reported data for codon recognition by cmo 5 U-containing (or mo 5 U-containing) leucine ( Nishiyama and Tokuda 2005 ; Sørensen et al. 2005 ) and serine ( Takai et al. 1999 ) tRNAs. The cmo 5 U-containing valine and alanine tRNAs can support growth of mutants lacking the corresponding G34-containing isoacceptors ( Tables 2 , ​ ,3), 3 ), although the growth rates of such mutants are reduced compared to a wild-type strain. At the other extreme is the cmo 5 U34-containing proline isoacceptor, which supports growth at a rate indistinguishable from a wild-type strain even when both the G34- and C34-containing proline isoacceptors are missing ( Näsvall et al. 2004 ). These dramatic differences between the different tRNAs could be the result of tRNAs having different relative efficiencies of recognizing one or more of their specific codons. Alternatively, the expression of the cmo 5 U34-containing tRNA Pro cmo5UGG could be high enough relative to the codon usage to allow efficient decoding even in the absence of the other proline tRNAs. However, among the cmo 5 U-containing alanine, valine, and proline tRNAs, the proline tRNA is the least abundant relative to the codon usage ( Table 4 ). Thus, codon usage and tRNA levels alone cannot explain the differences in phenotypes we observe.

Codon usage and tRNA availability

Comparing the relative rates of A-site selection for the different cmo 5 U34-containing proline, valine, and alanine tRNAs when they are the only isoacceptors present ( Fig. 5 , Δ valVW ; Fig. 7 , Δ alaXW ; Fig. 8 , Δ proKL ) the A-, G-, and U-ending codons were recognized at similar rates, while the rates of recognizing the C-ending alanine and valine codons are much lower (about fourfold lower than the other codons). In all cases, the rates at the A-ending codons were lower in the strains lacking the other isoacceptors than in the wild type. This was expected, as the remaining tRNAs have to read more of the codons that are normally also read by the other isoacceptors, leading to fewer tRNAs available to read the A-ending codon. In vitro a slightly lower rate of recognition was also observed toward the GCC (Ala) codon compared to the GCA (Ala) codon ( Kothe and Rodnina 2007 ). However, tRNA Pro cmo5UGG recognized the C-ending codon almost as efficiently as the other proline codons ( Fig. 8 ), which could partly explain why the mutant (Δ proL proK <> frt ) lacking the G34- and C34-containing proline isoacceptors has no apparent growth phenotype. One feature differentiating the anticodon loop in the cmo 5 U-containing proline tRNA from the anticodon loops in the other tested tRNAs is the presence of four consecutive purines (G35-G36-m 1 G37-A38). As purine–purine stacking is the most stable stacking interaction ( Saenger 1984 ), this may lead to an exceptionally stable anticodon loop through extensive stacking of these bases, perhaps contributing to the efficiency of decoding the CCC codon by tRNA Pro cmo5UGG .

Combining the lack of the G34-containing (and C34-containing) isoacceptors with hypo-modification (ho 5 U34 instead of cmo 5 U34) of the wobble nucleoside in the remaining isoacceptors also has different effects on the growth rates of the different mutants. All three mutants with only the cmo 5 U-containing isoacceptors left in the respective family codon boxes (Δ proL proK <> frt , Δ alaXW , and Δ valVW ) have significantly reduced growth rates when combined with a cmoB mutation ( Tables 1 , ​ ,3; 3 ; Näsvall et al. 2004 ), but the severity of the synergistic phenotypes is very different. The Δ alaXW cmoB2 mutant is virtually impossible to keep as a pure culture without accumulation of faster-growing suppressors ( Fig. 6 ). The nature of the suppressors that accumulated in the Δ alaXW cmoB2 and Δ valVW mutants was not examined in detail, but at least some of them segregated into small and large colonies, indicating that they may be amplifications of the tRNA genes (data not shown). As both mutants were suppressed by expressing more of the remaining tRNA ( Figs. 4 and ​ and6), 6 ), it is likely that amplification of the genes encoding these tRNAs would account for some of the suppressors. The effects of hypo-modification on the Δ proL proK <> frt and the Δ valVW mutants seem to be similar (although the Δ valVW mutant is very slow growing even in the presence of cmo 5 U34) ( Fig. 4 ). Comparing the effects of having ho 5 U in place of cmo 5 U on the A-site selection rate for the different tRNAs ( Figs. 5 , ​ ,7, 7 , ​ ,8, 8 , cf. LT2 and cmoB2 ; Fig. 8 , cf. Δ proKL and Δ proKL cmoB2 ), the only codons where we observed significant differences in all three family boxes are at the G-ending codons. The largest effect is at the G-ending alanine codon. Taken together, the data from the A-site selection assays indicate that the extreme phenotype of the cmoB2 Δ alaXW mutant probably is a combination of very poor recognition of the GCC codon due to the lack of the G34-containing alanine isoacceptor and the reduced efficiency of recognizing the GCG codon caused by the modification deficiency. In addition to the decreases in the selection rates at the G-ending codons, we also observed decreased rates on the C-ending valine and proline codons and the U-ending alanine and proline codons. The effect of cmo 5 U deficiency on the ability of the Δ proL proK <> frt and Δ valVW mutants to recognize their corresponding G-ending codons is not as large as for the Δ alaXW mutant, which could be an explanation of why the different mutants have such different growth phenotypes. There may be several reasons why cmo 5 U apparently is more important for the function of tRNA Ala cmo5UGC than it seems to be for the functions of tRNA Pro cmo5UGG and tRNA Val cmo5UAC . One might be that other features present in tRNA Pro cmo5UGG and tRNA Val cmo5UAC but absent in tRNA Ala cmo5UGC contribute to their efficient decoding. One candidate for such a feature would be the modification of position 37 (immediately 3′ of the anticodon). tRNA Pro has m 1 G37 ( Kuchino et al. 1984 ), and tRNA Val has m 6 A37 ( Kimura et al. 1971 ), while tRNA Ala has an unmodified adenosine at position 37 ( Lund and Dahlberg 1977 ). m 1 G37 has previously been demonstrated to be important for the A-site selection rate at all four proline codons ( Li et al. 1997 ). As the effects on growth rates of some of the mutants appear to be larger than one would expect from the changes in the A-site selection rates, it is possible that part of the growth phenotypes are due to defects in a stage of the translation elongation cycle other than the A-site selection. We have previously shown that having ho 5 U instead of cmo 5 U in tRNA Pro cmo5UGG actually leads to decreased +1 frameshifting at CCC codons ( Qian et al. 1998 ; Näsvall et al. 2004 ), which is why we do not think frameshifting in such a mutant reaches high enough levels to cause the observed growth-rate reduction.

It should be noted that there are organisms that have only a U34-containing tRNAs to read all four codons in some or all of the family codon boxes encoding Ser, Pro, Thr, Ala, Val, and Leu. Bacillus subtilis have only one proline tRNA (containing mo 5 U34) ( Yamada et al. 2005 ), while it has two (mo 5 U34 and G34) or three (mo 5 U34, G34, and C34) tRNAs in the other five boxes ( Sprinzl and Vassilenko 2005 ). Certain intracellular parasites, like the Mollicutes (to which the Mycoplasmas belong), have very small, A/T-rich genomes with a reduced number of tRNA genes compared to free-living bacteria. Some of these use only U34-containing tRNAs in all these boxes, while the others have additional G34-, C34-, or A34-containing tRNAs in some of the serine, threonine, alanine, valine, or leucine boxes (for review, see De Crécy-Lagard et al. 2007 ). The codon usages of these organisms are very strongly biased toward A- and U-ending codons. The modification status of tRNAs from most of these organisms are unknown, but at least Mycoplasma capricolum and Mycoplasma mycoides have unmodified uridines at the wobble position. This may indicate that during their reductive evolution, the Mollicutes (distantly related to Bacillus ) have lost the genes required to synthesize mo 5 U, and have unmodified U34. As these bacteria use U34-containing tRNAs to read primarily A- and U-ending codons, while they use C- and G-ending codons very rarely, this suggests that U34-containing tRNAs in the family codon boxes are unexpectedly efficient at reading U-ending codons, while this requirement may not be relevant for the rarely used G- and C-ending codons. This may be an exception enabled by special features of the ribosomes and/or tRNAs from these highly specialized organisms, but it may also be a general rule for U34-containing tRNAs in other organisms as well. In fact, completely unmodified E. coli tRNA Ser UGA (which normally has cmo 5 U34) is capable of reading UCA and UCU in an in vitro translation system, but UCG is very poorly translated unless the wobble nucleoside is modified to mo 5 U ( Takai et al. 1999 ). With this in mind, perhaps the unknown gene(s) encoding the enzyme(s) responsible for making ho 5 U34 from U34 in tRNA is essential for Salmonella and E. coli even in strains with the full complement of tRNAs, as the U34-containing alanine and valine tRNAs may require at least ho 5 U in order to recognize their G-ending codons efficiently enough to support growth.

Relevant to the coding capacities in Mollicutes is the “two out of three” decoding model, which states that the third position of the codon–anticodon interaction can be disregarded, as long as the interactions in the first two positions are strong ( Lagerkvist 1978 ). This would apply to codons where the first two positions form G-C pairs (Pro, Ala, Arg, and Gly) but not to codons forming only A-U pairs (such as the Phe/Leu, Ile/Met, Tyr/Stop, and Asn/Lys mixed codon boxes). The two out of three model is supported by in vitro translation experiments in which the E. coli cmo 5 U-containing alanine and valine tRNAs could incorporate the respective cognate amino acids at all four of their codons even in the presence of a competing cognate tRNA ( Mitra et al. 1979 ; Samuelsson et al. 1980 ). The relative efficiency of the cmo 5 U-containing tRNA in reading the four alanine and valine codons in the presence of the cognate tRNA is similar to our results obtained using the A-site selection assay in cells with only the fully modified cmo 5 U-containing tRNA present to read all four codons ( Figs. 5 , ​ ,7). 7 ). Lagerkvist assumed that the efficient reading of U-ending codons was caused by the presence of cmo 5 U, while the less efficient reading of C-ending codons was regarded as two-out-of-three reading that did not involve an interaction at the third position. However, in vivo ( Figs. 5 , ​ ,7, 7 , ​ ,8) 8 ) the hypo-modified derivatives were less efficient to read the G-ending and sometimes the U- or C-ending codons, although the extent of the reductions at the C- and U-ending codons may be masked by the presence of the competing G34-containing tRNAs. Thus, the modification of the wobble base may contribute to the decoding efficiency even at C-ending codons and cannot be disregarded as suggested by the two out of three model. However, as we have no mutant that has unmodified U34, we cannot conclusively say if alanine, valine, or proline tRNA in such a mutant would still read U- or C-ending codons. Moreover, the fact that the Leu and Thr family boxes cannot be decoded by a single tRNA ( Table 1 ; Nishiyama and Tokuda 2005 ; Sørensen et al. 2005 ) is not consistent with the two out of three decoding model. Why the cmo 5 U-containing Ala and Pro tRNAs can read C-ending codons but the Leu, Thr, and Ser tRNAs cannot may be related to the stability of the first two base pairs in the codon–anticodon complex (Ala and Pro make two G-C pairs, whereas Leu, Thr, and Ser make only one G-C pair). Still, it is unclear why tRNA Val cmo5UAC can read all four codons (although not efficiently enough to support normal growth) when it also makes only one G-C pair ( Fig. 5 ; Table 2 ).

In his wobble hypothesis, Crick predicted that U34 in tRNA would be able to form a wobble pair with G(III) in the mRNA ( Crick 1966 ), with two hydrogen bonds between U34 and G(III). In order to allow formation of such a base pair, either U34 has to be displaced toward the major groove or G(III) has to be displaced toward the minor groove of the codon–anticodon mini-helix (a move of ∼2.5 Å for one of the glycosidic bonds compared to a Watson–Crick base pair) ( Fig. 9B ; Crick 1966 ). It is unlikely that G(III) would be allowed to move, since its movement is restricted by its interactions with residues in the ribosomal 30S subunit ( Ogle et al. 2001 ; Murphy et al. 2004 ). If U34 would move the entire distance required to form a U-G wobble pair, it would be unable to make a stacking interaction with the base in position 35 of the tRNA ( Murphy et al. 2004 ), which could perhaps lead to poor recognition of G-ending codons by tRNAs with unmodified U34. Weixlbaumer et al. (2007) have recently solved the crystal structures of Thermus thermophilus 70S ribosomes containing an ASL Val cmo5UAC (with the modifications cmo 5 U34 and m 6 A37) bound to the four Val codons in the A-site. One interesting feature of the cmo 5 U-G base pair is that it has standard Watson–Crick geometry rather than the expected wobble geometry ( Fig. 9 ). This means that the 4-carbonyl group of cmo 5 U34 has to be in the rare enol form rather than the normal keto form and that the base pair makes three hydrogen bonds ( Fig. 9B ). Hillen et al. (1978) showed that mo 5 U has a shifted keto–enol equilibrium compared to uridine and ho 5 U. Our data showing that tRNAs with cmo 5 U34 are significantly more efficient than tRNAs with ho 5 U at reading G-ending codons are consistent with the observed crystal structure and predict that cmo 5 U34 is, indeed, in the enol form. In all four of these structures, the ether oxygen (O5) of the modification forms a hydrogen bond to the 2′-OH of U33 ( Fig. 9A ). This leads to a more constrained anticodon than if the wobble uridine would have been unmodified ( Weixlbaumer et al. 2007 ), with the wobble nucleoside “locked” in position close to where it would be in a Watson–Crick base pair. The cmo 5 U-U and cmo 5 U-C pairs in the structures only form one hydrogen bond between the codon and anticodon bases ( Weixlbaumer et al. 2007 ). Part of the stabilization needed to allow these pairs may come from the observed hydrogen bond between the O5 of cmo 5 U34 and the 2′-OH of U33. The cmo 5 U-U pair may be further stabilized by interactions with 16S rRNA, whereas the cmo 5 U-C pair may be slightly destabilized by poor stacking between C(III) and its 5′-base ( Weixlbaumer et al. 2007 ). These differences between the cmo 5 U-U and cmo 5 U-C pairs may explain why the C-ending codons are poorly recognized by the cmo 5 U-containing alanine and valine tRNAs, while the U-ending codons are recognized efficiently ( Figs. 5 , ​ ,7). 7 ). Weixlbaumer et al. also point out that the carboxyl group of cmo 5 U34 is close enough to the oxygen of the 4-carbonyl group of U(III) to be able to form a hydrogen bond if the carboxyl group is protonated ( Weixlbaumer et al. 2007 ). This would provide even further stability to the cmo 5 U-U pair but not to the cmo 5 U-C pair. In such a case, the hypo-modified ho 5 U34-containing tRNAs in a cmoB mutant (lacking the carboxyl group) would be affected in their reading of U-ending codons. Since we see no large effect on the rates of reading U-ending codons, our data do not support this suggestion. Another important observation in these structures is that in all four pairs, the ribose of cmo 5 U34 adopts the 3′-endo conformation, and the modification does not interact with its 5′-phosphate as predicted by Yokoyama et al. (1985) .

( A ) The cmo 5 U34-G(III) base pair seen in the crystal structure solved by Weixlbaumer et al. (2007) (Protein Databank accession code 2UU9). The hydrogen bond between the 2′-OH of U33 and the O5 ether oxygen of cmo 5 U34 is indicated. ( B ) Keto-enol tautomerization of cmo 5 U. ( Left ) The keto tautomer of cmo 5 U (black), forming a wobble pair with G (gray) as predicted by Crick. ( Right ) The enol tautomer of cmo 5 U (black), engaged in a base pair with G (gray). The geometry of the cmo 5 U-G pair is the same as for a Watson–Crick (A-U or G-C) base pair.

Based on the structures discussed above ( Fig. 9 ; Weixlbaumer et al. 2007 ) and our in vivo data, we suggest that the function of cmo 5 U34 (and mo 5 U34) may be dual: Firstly, the ether oxygen (or the hydroxyl of ho 5 U) stabilizes the anticodon loop in a conformation where the wobble uridine is locked in position to form a base pair with Watson–Crick geometry. This leads to sufficient stabilization to compensate for the poor interactions between cmo 5 U34 and U(III) or C(III). Secondly, the rest of the modification (–CH 3 in mo 5 U or –CH 3 COOH in cmo 5 U) stabilizes the enol form of the wobble nucleoside, promoting a nonstandard U34-G(III) base pair.

MATERIALS AND METHODS

Bacteria and growth conditions.

The strains and plasmids used are listed in Table 5 . All Salmonella strains are derivatives of Salmonella enterica serovar Typhimurium strain LT2. As solid rich medium, TYS (10 g of Trypticase peptone, 5 g of yeast extract, 5 g of NaCl, and 15 g of agar per liter) was used. As solid minimal medium, medium E ( Vogel and Bonner 1956 ) containing 15 g of agar per liter and 0.2% glucose was used. As rich liquid medium, either LB or NAA (0.8% Difco nutrient broth; Difco Laboratories) supplemented with the aromatic amino acids, aromatic vitamins, and adenine at concentrations as described previously ( Davis et al. 1980 ) was used. For growth rate determination and assay of β-galactosidase activities, Rich MOPS ( Neidhardt et al. 1977 ) was used. All growth was done at 37°C. Antibiotics were used at the following concentrations: Carbenicillin (Cb): 50 mg/L; Kanamycin (Km): 100 mg/L; Chloramphenicol (Cm): 12.5 mg/L; Tetracycline (Tet): 15 mg/L.

Salmonella enterica serovar Typhimurium and Escherichia coli strains and plasmids

Genetic procedures

To transfer chromosomal markers or plasmids between Salmonella strains, transductions were performed as described previously ( Davis et al. 1980 ) with a derivative of phage P22 containing the mutations HT105/I ( Schmieger 1972 ) and int-201 ( Scott et al. 1975 ). Green indicator plates ( Chan et al. 1972 ) were used for testing that the clones were phage free and phage sensitive.

Molecular cloning procedures

PCR fragments were purified from agarose gels using Wizard DNA clean-up resin (Promega) or directly using PCR kleen-spin (Bio-Rad). DNA sequencing was done using the DYEnamic ET terminator cycle sequencing kit (Amersham Pharmacia Biotech Inc.) or the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems).

A 1.8-kb crossover PCR product containing the deletion of the alaXW operon (from 101 nt upstream to 55 nt downstream of alaXW ) was generated as described by Link et al. (1997) and cloned into the suicide vector pDM4 ( Milton et al. 1996 ). The plasmid (pUST300) was transformed into E. coli strain GRB1371, and the resulting strain (GRB1748) was used as donor in conjugation with the Salmonella strain GT1796 as recipient. Cointegrates were segregated by nonselective growth in LB supplemented with 5% sucrose. Sucrose-resistant segregants were screened by PCR to find clones carrying the alaXW deletion. To generate the valVW deletion, a 1.2-kb crossover fragment containing a deletion of the valV and valW genes (from 6 nt upstream of valV to 28 nt downstream of valW ) was cloned into the temperature-sensitive suicide vector pMAK705 ( Hamilton et al. 1989 ), generating pUST301. Cointegrates were obtained by growing at 44°C in the presence of chloramphenicol and segregated by growing nonselectively at 30°C. Chloramphenicol-sensitive segregants were screened by PCR to find clones carrying the valVW deletion on the chromosome.

We used the method described by Datsenko and Wanner (2000) to replace thrT , thrV , or thrW in strain GT6315 (an LT2 derivative with the λ-red recombinase plasmid pKD46) with the kanamycin resistance cassette amplified from plasmid pKD4 ( Datsenko and Wanner 2000 ). The <> kan alleles were transferred by P22 transductions into strain LT2 before the FLP-recombinase helper plasmid pCP20 ( Datsenko and Wanner 2000 ) was introduced to convert the <> kan alleles into <> frt alleles. To combine the different mutations, we used P22 lysates grown on strains containing the different thr <> kan alleles as donors and cultures of strains containing the different thr <> frt alleles as recipients in transductions, selecting Km R transductants ( Table 1 ). To examine the presence of duplications in the rare transductants that appeared when trying to construct a thrT , thrV double mutant, 37 transductants (from transductions using thrT <> kan as donor and thrV <> frt as recipient), including clones that appeared after up to 72 h of incubation, were checked by PCR reactions designed to distinguish between the wild-type and thrT <> kan alleles.

The cmoB2 <> cat allele was constructed to be identical to the cmoB2 <> kan allele ( Näsvall et al. 2004 ), except the chloramphenicol resistance cassette from plasmid pKD3 was used instead of the kanamycin resistance cassette from pKD4.

Nomenclature of mutants

An allele number followed by <> and “ kan ” or “ cat ” indicates that the gene is replaced by the kanamycin or chloramphenicol resistance cassettes from plasmid pKD4 or pKD3, respectively (e.g., cmoB2 <> kan or cmoB2 <> cat ). After FLP-recombinase-mediated removal of the cassette, the mutation is referred to with the same allele number, but with “ frt ” as description of the resulting scar sequence (e.g., cmoB2 <> frt ). The same allele number is also used for replacements where removal of two different FRT (Flp Recombinase Target sequence) flanked antibiotic resistance cassettes would produce an identical end result; hence cmoB2 <> kan , cmoB2 <> cat , and cmoB2 <> frt have the same allele number. A “Δ” before a gene name refers to a precise deletion rather than a replacement (e.g., “Δ proL ”).

Nomenclature of tRNAs

In most cases, tRNAs are referred to with their cognate amino acid in three-letter code in superscript and with their anticodon sequence (5′→3′) in subscript, i.e., tRNA Thr cmo5UGC refers to a threonine tRNA with the anticodon cmo 5 U34-G35-U36. In some cases, the names of tRNA genes are followed by the wobble nucleoside of the corresponding tRNA within parentheses.

Determination of growth rates

Overnight cultures of the different strains were diluted to ∼0.05–0.1 OD 420 units in pre-warmed medium and were pre-grown to OD 420 ≈1.0. The cultures were then diluted to OD 420 ≈0.02–0.06, and growth was monitored with a Shimadzu UV-1601 spectrophotometer at 420 nm. When the OD of the cultures reached OD 420 ≈1.0, they were again diluted into fresh pre-warmed medium. The cultures were judged to be in balanced growth when the growth rate determined after a dilution did not change more than 5% from what it was before dilution. We did not wait for the slow-growing Δ valVW (GT6300) or Δ valVW , cmoB2 (GT6972) mutants ( Table 2 ) to reach balanced growth; instead, they were treated as follows: Overnight cultures of GT6300 or still growing cultures (with an OD 420 below 1.0) of GT6972 were diluted once to OD 420 ≈0.05, and growth was monitored until the OD again reached 1.0. Cultures of GT6300 were diluted once more, and monitoring continued until OD 420 ≈1.0. Samples were withdrawn from the cultures and plated to estimate the proportion of faster-growing suppressor mutants. The data in Table 2 were calculated from cultures that contained <5% suppressors at the end of the experiments. The specific growth rate is expressed as k (h −1 ), where k =ln 2/ g (where g is the generation time in hours). Strain LT2 (wild-type) was always grown in parallel with the mutants to get a reference value for each experiment. Because of this, the values for LT2 in Tables 2 and ​ and3 3 differ slightly from each other. To estimate the colony diameters reported in the legends for Figure 4 and Figure 6 , the plates were photographed with a ChemiDoc XRS (Bio-Rad). Quantity One software (Bio-Rad) was used to measure the approximate width and height of square boxes manually fitted to surround each colony. The values are averages of five representative colonies (except the cmoB2 <> kan mutant in Fig. 6B , sector 2, where only two colonies were sufficiently separated from other colonies). The average diameter of strain LT2 (containing the relevant plasmids) growing on the same plate was set to 1.0.

Determination of A-site selection rates

Strains were grown overnight at 37°C in Rich MOPS medium supplemented with 12.5 μg/mL chloramphenicol, subcultured and grown to mid-log phase (OD 600 ≈0.5 using a Shimadzu UV-1201 spectrophotometer, corresponding to ∼2 × 10 8 CFU/mL). β-Galactosidase activity was measured as described by Miller (1972) (using the alternative method with chloroform and SDS instead of toluene to open the cells). ONPG was from Sigma. For each strain to be tested, at least two independent cultures were grown, and each strain was tested in at least three separate experiments. The plasmids for testing the alanine and valine codons were constructed by mutagenizing previously constructed plasmids ( Curran and Yarus 1989 ) according to the protocol for the QuikChange Site-Directed Mutagenesis kit (Stratagene). The numbers presented in Figures 5 , ​ ,7, 7 , and ​ and8 8 are the A-site selection rates expressed as

where F is the frequency of frameshifting, determined by dividing the β-galactosidase activity of the test codon constructs with that of the pseudo-wild-type plasmid pJC27.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Cancer Foundation (Project 680) and the Swedish Science Research Council (Project BU-2930). We are grateful to Drs. V. Ramakrishnan and A. Weixlbaumer (Cambridge, UK) for making their manuscript available prior to its publication and for the kind gift of the unpublished Figure 9A . We thank Gunilla Jäger for performing β-galactosidase assays and Tord Hagervall, Anders Byström, and Anders Esberg for critical reading of the manuscript.

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.731007 .

• Chan, R.K., Botstein, D., Watanabe, T., Ogata, Y. Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium . II. Properties of a high-frequency-transducing lysate. Virology. 1972; 50 :883–898. [ PubMed ] [ Google Scholar ]
• Crick, F.H.C. Codon–anticodon pairing. The wobble hypothesis. J. Mol. Biol. 1966; 19 :548–555. [ PubMed ] [ Google Scholar ]
• Curran, J.F., Yarus, M. Rates of aminoacyl-tRNA selection at 29 sense codons in vivo. J. Mol. Biol. 1989; 209 :65–77. [ PubMed ] [ Google Scholar ]
• Datsenko, K.A., Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. 2000; 97 :6640–6645. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Davis, W., Botstein, D., Roth, J.R. A manual for genetic engineering: Advanced bacterial genetics. Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1980. [ Google Scholar ]
• De Crécy-Lagard, V., Marck, C., Brochier-Armanet, C., Grosjean, H. Comparative RNomics and Modomics in mollicutes: Prediction of gene function and evolutionary implications. IUBMB Life. 2007; 59 :634–658. [ PubMed ] [ Google Scholar ]
• Dong, H.J., Nilsson, L., Kurland, C.G. Covariation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J. Mol. Biol. 1996; 260 :649–663. [ PubMed ] [ Google Scholar ]
• Gabriel, K., Schneider, J., McClain, W.H. Functional evidence for indirect recognition of G•U in tRNA Ala by alanyl-tRNA synthetase. Science. 1996; 271 :195–197. [ PubMed ] [ Google Scholar ]
• Hamilton, C.M., Aldea, M., Washburn, B.K., Babitzke, P., Kushner, S.R. New method for generating deletions and gene replacements in Escherichia coli . J. Bacteriol. 1989; 171 :4617–4622. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Hillen, W., Egert, E., Lindner, H.J., Gassen, H.G., Vorbruggen, H. 5-Methoxyuridine: The influence of 5-substituents on the Keto-enol tautomerism of the 4-carbonyl group. J. Carbohydrates Nucleosides Nucleotides. 1978; 5 :23–32. [ Google Scholar ]
• Ishikura, H., Yamada, Y., Nishimura, S. Structure of serine tRNA from Escherichia coli . I. Purification of serine tRNA's with different codon responses. Biochim. Biophys. Acta. 1971; 228 :471–481. [ PubMed ] [ Google Scholar ]
• Kimura, F., Harada, F., Nishimura, S. Primary sequence of tRNA-Val-1 from Escherichia coli B. II. Isolation of large fragments by limited digestion with RNases, and overlapping of fragments to reduce the total primary sequence. Biochemistry. 1971; 10 :3277–3283. [ PubMed ] [ Google Scholar ]
• Kothe, U., Rodnina, M.V. Codon reading by tRNA Ala with modified uridine in the wobble position. Mol. Cell. 2007; 25 :167–174. [ PubMed ] [ Google Scholar ]
• Kuchino, Y., Yabusaki, Y., Mori, F., Nishimura, S. Nucleotide sequences of three proline tRNAs from Salmonella typhimurium . Nucleic Acids Res. 1984; 12 :1559–1562. doi: 10.1093/nar/12.3.1559. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lagerkvist, U. “Two out of three”: An alternative method for codon reading. Proc. Natl. Acad. Sci. 1978; 75 :1759–1762. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Li, J.N., Esberg, B., Curran, J.F., Björk, G.R. Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner. J. Mol. Biol. 1997; 271 :209–221. [ PubMed ] [ Google Scholar ]
• Link, A.J., Phillips, D., Church, G.M. Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli : Application to open reading frame characterization. J. Bacteriol. 1997; 179 :6228–6237. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Lund, E., Dahlberg, J.E. Spacer transfer RNAs in ribosomal RNA transcripts of E. coli : Processing of 30S ribosomal RNA in vitro. Cell. 1977; 11 :247–262. [ PubMed ] [ Google Scholar ]
• Miller, J.H. Experiments in molecular genetics. Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1972. [ Google Scholar ]
• Milton, D.L., O'Toole, R., Horstedt, P., Wolf-Watz, H. Flagellin A is essential for the virulence of Vibrio anguillarum . J. Bacteriol. 1996; 178 :1310–1319. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Mitra, S.K., Lustig, F., Akesson, B., Axberg, T., Elias, P., Lagerkvist, U. Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro. J. Biol. Chem. 1979; 254 :6397–6401. [ PubMed ] [ Google Scholar ]
• Murphy, F.V., Ramakrishnan, V., Malkiewicz, A., Agris, P.F. The role of modifications in codon discrimination by tRNA Lys UUU . Nat. Struct. Mol. Biol. 2004; 11 :1186–1192. [ PubMed ] [ Google Scholar ]
• Nakamura, Y., Gojobori, T., Ikemura, T. Codon usage tabulated from international DNA sequence databases: Status for the year 2000. Nucleic Acids Res. 2000; 28 :292. doi: 10.1093/nar/28.1.292. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Näsvall, S.J., Chen, P., Björk, G.R. The modified wobble nucleoside uridine-5-oxyacetic acid in tRNA Pro cmo5UGG promotes reading of all four proline codons in vivo. RNA. 2004; 10 :1662–1673. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Neidhardt, F.C., Bloch, P.L., Pedersen, S., Reeh, S. Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli . J. Bacteriol. 1977; 129 :378–387. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Nishiyama, K.I., Tokuda, H. Genes coding for SecG and Leu2-tRNA form an operon to give an unusual RNA comprising mRNA and a tRNA precursor. Biochim. Biophys. Acta. 2005; 1729 :166–173. [ PubMed ] [ Google Scholar ]
• O'Connor, M. Insertions in the anticodon loop of tRNA Gln 1 ( sufG ) and tRNA Lys promote quadruplet decoding of CAAA. Nucleic Acids Res. 2002; 30 :1985–1990. doi: 10.1093/nar/30.9.1985. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Oda, K., Kimura, F., Harada, F., Nishimura, S. Restoration of valine acceptor activity by combining oligonucleotide fragments derived from a Bacillus subtilis ribonuclease digest of Escherichia coli valine transfer RNA. Biochim. Biophys. Acta. 1969; 179 :97–105. [ PubMed ] [ Google Scholar ]
• Ogle, J.M., Brodersen, D.E., Clemons W.M., Jr, Tarry, M.J., Carter, A.P., Ramakrishnan, V. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science. 2001; 292 :897–902. [ PubMed ] [ Google Scholar ]
• Phelps, S.S., Malkiewicz, A., Agris, P.F., Joseph, S. Modified nucleotides in tRNA Lys and tRNA Val are important for translocation. J. Mol. Biol. 2004; 338 :439–444. [ PubMed ] [ Google Scholar ]
• Qian, Q., Li, J.N., Zhao, H., Hagervall, T.G., Farabaugh, P.J., Björk, G.R. A new model for phenotypic suppression of frameshift mutations by mutant tRNAs. Mol. Cell. 1998; 1 :471–482. [ PubMed ] [ Google Scholar ]
• Saenger, W. 6.6 Thermodynamic description of stacking interactions. Principles of nucleic acid structure. Springer-Verlag; New York: 1984. pp. 134–137. [ Google Scholar ]
• Samuelsson, T., Elias, P., Lustig, F., Axberg, T., Fölsch, G., Åkesson, B., Lagerkvist, U. Aberrations of the classic codon reading scheme during protein synthesis in vitro. J. Biol. Chem. 1980; 255 :4583–4588. [ PubMed ] [ Google Scholar ]
• Schmieger, H. Phage P22 -mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 1972; 119 :75–88. [ PubMed ] [ Google Scholar ]
• Scott, J.F., Roth, J.R., Artz, S.W. Regulation of histidine operon does not require hisG enzyme. Proc. Natl. Acad. Sci. 1975; 72 :5021–5025. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Simon, R., Priefer, U., Pühler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology (N. Y.) 1983; 1 :784–791. [ Google Scholar ]
• Sørensen, M.A., Elf, J., Bouakaz, E., Tenson, T., Sanyal, S., Björk, G.R., Ehrenberg, M. Overexpression of a tRNA Leu isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading. J. Mol. Biol. 2005; 354 :16–24. [ PubMed ] [ Google Scholar ]
• Sprinzl, M., Vassilenko, K.S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 2005; 33 :D139–D140. doi: 10.1093/nar/gki012. [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Stoker, N.G., Fairweather, N.F., Spratt, B.G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli . Gene. 1982; 18 :335–341. [ PubMed ] [ Google Scholar ]
• Takai, K., Okumura, S., Hosono, K., Yokoyama, S., Takaku, H. A single uridine modification at the wobble position of an artificial tRNA enhances wobbling in an Escherichia coli cell-free translation system. FEBS Lett. 1999; 447 :1–4. [ PubMed ] [ Google Scholar ]
• Vila-Sanjurjo, A., Squires, C.L., Dahlberg, A.E. Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli . J. Mol. Biol. 1999; 293 :1–8. [ PubMed ] [ Google Scholar ]
• Vogel, H.J., Bonner, D.M. Acetylornithinase of Escherichia coli : Partial purification and some properties. J. Biol. Chem. 1956; 218 :97–106. [ PubMed ] [ Google Scholar ]
• Weixlbaumer, A., Murphy, F.V., Dziergowska, A., Malkiewicz, A., Vendeix, F.A., Agris, P.F., Ramakrishnan, V. Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines. Nat. Struct. Mol. Biol. 2007; 14 :498–502. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Yamada, Y., Matsugi, J., Ishikura, H., Murao, K. Bacillus subtilis tRNA Pro with the anticodon mo5UGG can recognize the codon CCC. Biochim. Biophys. Acta. 2005; 1728 :143–149. [ PubMed ] [ Google Scholar ]
• Yokoyama, S., Watanabe, T., Murao, K., Ishikura, H., Yamaizumi, Z., Nishimura, S., Miyazawa, T. Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon. Proc. Natl. Acad. Sci. 1985; 82 :4905–4909. [ PMC free article ] [ PubMed ] [ Google Scholar ]

Celebrating wobble decoding: Half a century and still much is new

Affiliations.

• 1 a The RNA Institute , State University of New York , Albany , NY , USA.
• 2 b Department of Biology , State University of New York , Albany , NY , USA.
• 3 c Department of Chemistry , State University of New York , Albany , NY , USA.
• PMID: 28812932
• PMCID: PMC6103715
• DOI: 10.1080/15476286.2017.1356562

A simple post-transcriptional modification of tRNA, deamination of adenosine to inosine at the first, or wobble, position of the anticodon, inspired Francis Crick's Wobble Hypothesis 50 years ago. Many more naturally-occurring modifications have been elucidated and continue to be discovered. The post-transcriptional modifications of tRNA's anticodon domain are the most diverse and chemically complex of any RNA modifications. Their contribution with regards to chemistry, structure and dynamics reveal individual and combined effects on tRNA function in recognition of cognate and wobble codons. As forecast by the Modified Wobble Hypothesis 25 years ago, some individual modifications at tRNA's wobble position have evolved to restrict codon recognition whereas others expand the tRNA's ability to read as many as four synonymous codons. Here, we review tRNA wobble codon recognition using specific examples of simple and complex modification chemistries that alter tRNA function. Understanding natural modifications has inspired evolutionary insights and possible innovation in protein synthesis.

Keywords: Modified Wobble Hypothesis; Wobble Hypothesis; cognate and wobble codon recognition; modified nucleosides; nucleoside tautomers; tRNA; translation; wobble decoding; wobble nucleoside.

Publication types

• Research Support, N.I.H., Extramural
• Research Support, U.S. Gov't, Non-P.H.S.
• Adenosine / genetics
• Adenosine / metabolism*
• Archaea / genetics
• Archaea / metabolism
• Bacteria / genetics
• Bacteria / metabolism
• Base Pairing
• Deamination
• Eukaryota / genetics
• Eukaryota / metabolism
• Evolution, Molecular
• Genetic Code*
• Inosine / genetics
• Inosine / metabolism*
• Models, Molecular
• Nucleic Acid Conformation
• Protein Biosynthesis*
• RNA Processing, Post-Transcriptional*
• RNA, Transfer / chemistry*
• RNA, Transfer / genetics
• RNA, Transfer / metabolism
• RNA, Transfer

Grants and funding

• R01 GM110588/GM/NIGMS NIH HHS/United States

Wobble hypothesis

wobble hypothesis (Science: molecular biology) explains why the base inosine is included in position 1 in the anticodons of various t rNAs, why many mRNA codon words translate to a single amino acid , why there are appreciably fewer t RNAs than mRNA codon types and why the redundant nature of the genetic code translates into a precise set of 20 amino acids. inosine in position 1 in the anticodon can base pair with A, u or C in position 3 in the mRNA codon , so that for example UCU, UCC, UCA all code for serine using an inosine anticodon.

Last updated on July 24th, 2022

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A theory to explain the partial degeneracy of the genetic code due to the fact that some t-RNA molecules can recognize more than one codon. The theory proposes that the first two bases in the codon and anticodon will form complementary pairs in the normal antiparallel fashion. However, a degree of steric freedom or ‘wobble’ is allowed in the base-pairing at the third position. Thus, for serine, six m-RNA codons may be paired with only three t-RNA anticodons.

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