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

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 (cmo⁵U34) 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 cmo⁵U-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 cmo⁵U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo⁵U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo⁵U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo⁵U 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.     

Codon reading by tRNAAla with modified uridine in the wobble position

tRNAs reading four-codon families often have a modified uridine, cmo(5)U(34), at the wobble position of the anticodon. Here, we examine the effects on the decoding mechanism of a cmo(5)U modification in tRNA(1B)(Ala), anticodon C(36)G(35)cmo(5)U(34). tRNA(1B)(Ala) reads its cognate codons in a manner that is very similar to that of tRNA(Phe). As Ala codons are GC rich and Phe codons AU rich, this similarity suggests a uniform decoding mechanism that is independent of the GC content of the codon-anticodon duplex or the identity of the tRNA. The presence of cmo(5)U at the wobble position of tRNA(1B)(Ala) permits fairly efficient reading of non-Watson-Crick and nonwobble bases in the third codon position, e.g., the GCC codon. The ribosome accepts the C-cmo(5)U pair as an almost-correct base pair, unlike third-position mismatches, which lead to the incorporation of incorrect amino acids and are efficiently rejected.     

Accurate translation of the genetic code depends on tRNA modified nucleosides

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U(34) to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s(2)U(34)), 5-methylaminomethyluridine at position 34 (mnm(5)U(34)), and 6-threonylcarbamoyladenosine at position 37 (t(6)A(37)) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA(UUU)(Lys) at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.     

In vitro codon-reading specificities of unmodified tRNA molecules with different anticodons on the sequence background of Escherichia coli tRNASer

The codon-reading properties of wobble-position variants of the unmodified form of Escherichia coli tRNASer1 (the UGA anticodon) were measured in a cell-free translation system. Two variants, with the AGA and CGA anticodons, each exclusively read a single codon, UCU and UCG, respectively. The only case of efficient wobbling occurred with the variant with the GGA anticodon, which reads the UCU codon in addition to the UCC codon. Surprisingly, this wobble reading is more efficient than the Watson-Crick reading by the variant with the AGA anticodon. Furthermore, we prepared tRNA variants with AA, UC, and CU, instead of GA, in the second and third positions and measured their relative efficiencies in the reading of codons starting with UU, GA, and AG, respectively. The specificity concerning the wobble position is essentially the same as that in the case of the codons starting with UC.     

Identification and codon reading properties of 5-cyanomethyl uridine,a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA₂^Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA₂^Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.     

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.     

Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.     

Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA₂^Ile to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA^Ile-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA₂^Ile is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA₁^Ile, in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.     

Exceptional codon recognition by the glutamine tRNAs in Saccharomyces cerevisiae.

Recently, it was shown that wild-type glutamine tRNAs in yeast cause low-level nonsense suppression that can be enhanced by increasing glutamine tRNA gene copy number. In order to investigate glutamine tRNA behavior further, anticodon mutations that confer nonsense suppression were identified in yeast sup70 gene, which codes for glutamine tRNA(CAG). In this study we show that suppressors derived by mutation severely limit growth such that suppressor-bearing spores germinate but arrest cell division at approximately the 50 cell stage. Analysis of a sup70 deletion was used to establish that growth limitation results from loss of wild-type glutamine tRNA(CAG) function. By exploiting the growth inhibition of sup70 alleles, some exceptional codon recognition properties of glutamine tRNAs were revealed. Our results indicate that amber suppressor glutamine tRNA(UAG) can translate 5'-CAG-3' glutamine codons with low efficiency in the presence of an A/C mismatch at the first position of the codon, suggesting that reading may occur at a low level by a two-out-of-three reading mechanism. In addition, when glutamine tRNA(CAA) is over-expressed in vivo, it translates 5'-CAG-3' codons using a mechanism that resembles prokaryotic-like U/G wobble, which normally does not occur in yeast. Our studies also suggest that the yeast glutamine tRNA suppressors could potentially be exploited to express ciliated protozoan genes that normally contain internal 5'-UAG-3' and 5'-UAA-3' codons.     

Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.

Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.     

The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes

In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), 5-methoxycarbonylmethyluridine (mcm⁵U), 5-carbamoylmethyluridine (ncm⁵U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm⁵Um) nucleosides in the anticodon at the wobble position (U₃₄). Earlier we showed that mutants unable to form the side chain at position 5 (ncm⁵ or mcm⁵) or lacking sulphur at position 2 (s²) of U₃₄ result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm⁵ and s² side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.     

The role of modifications in codon discrimination by tRNA(Lys)UUU

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.     

Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs

There exists a similarity among the synonymous codon choice patterns of the yeast nuclear genes that have been sequenced thus far although these genes encode different types of protein molecules, and the patterns are significantly different from those of Escherichia coli genes. Based on constraints caused by the availability of E. coli transfer RNAs and the nature of their codon recognition related to the modified nucleotides at the anticodon wobble position, the characteristic patterns of synonymous codon choice commonly found for E. coli genes have been almost completely explained (Ikemura, 1981a, b). In the present paper, tRNAs of the yeast Saccharomyces cerevisiae were separated by two-dimensional polyacrylamide gel electrophoresis and the relative abundance of purified tRNA molecules was measured on the basis of molecular numbers in cells. A strong correlation between tRNA abundance and codon choice was found for each nuclear gene of yeast, but the correlation was less significant for 2μ plasmid genes. According to the criteria proposed for E. coli genes (Ikemura, 1981b) the order of codon preference in yeast nuclear genes was predicted based on the abundance of yeast isoaccepting tRNAs and on the nature of the modified nucleotides at their anticodons. Clear correlations between predictions and the actual preferences among synonymous codons were revealed, indicating that the codon choices in yeast genes are also constrained by a combination of tRNA availability and nature of its codon recognition. Then the difference in synonymous codon use between the two organisms can be attributed to the difference in these two factors.     

Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.     

Mutation and selection on the wobble nucleotide in tRNA anticodons in marine bivalve mitochondrial genomes

Background

Animal mitochondrial genomes typically encode one tRNA for each synonymous codon family, so that each tRNA anticodon essentially has to wobble to recognize two or four synonymous codons. Several factors have been hypothesized to determine the nucleotide at the wobble site of a tRNA anticodon in mitochondrial genomes, such as the codon-anticodon adaptation hypothesis, the wobble versatility hypothesis, the translation initiation and elongation conflict hypothesis, and the wobble cost hypothesis.

Principal Findings

In this study, we analyzed codon usage and tRNA anticodon wobble sites of 29 marine bivalve mitochondrial genomes to evaluate features of the wobble nucleotides in tRNA anticodons. The strand-specific mutation bias favors G and T on the H strand in all the 29 marine bivalve mitochondrial genomes. A bias favoring G and T is also visible in the third codon positions of protein-coding genes and the wobble sites of anticodons, rejecting that codon usage bias drives the wobble sites of tRNA anticodons or tRNA anticodon bias drives the evolution of codon usage. Almost all codon families (98.9%) from marine bivalve mitogenomes support the wobble versatility hypothesis. There are a few interesting exceptions involving tRNA^Trp with an anticodon CCA fixed in Pectinoida species, tRNA^Ser with a GCU anticodon fixed in Mytiloida mitogenomes, and the uniform anticodon CAU of tRNA^Met translating the AUR codon family.

Conclusions/Significance

These results demonstrate that most of the nucleotides at the wobble sites of tRNA anticodons in marine bivalve mitogenomes are determined by wobble versatility. Other factors such as the translation initiation and elongation conflict, and the cost of wobble translation may contribute to the determination of the wobble nucleotide in tRNA anticodons. The finding presented here provides valuable insights into the previous hypotheses of the wobble nucleotide in tRNA anticodons by adding some new evidence.     

Deciphering synonymous codons in the three domains of life: Co-evolution with specific tRNA modification enzymes

The strategies organisms use to decode synonymous codons in cytosolic protein synthesis are not uniform. The complete isoacceptor tRNA repertoire and the type of modified nucleoside found at the wobble position 34 of their anticodons were analyzed in all kingdoms of life. This led to the identification of four main decoding strategies that are diversely used in Bacteria, Archaea and Eukarya. Many of the modern tRNA modification enzymes acting at position 34 of tRNAs are present only in specific domains and obviously have arisen late during evolution. In an evolutionary fine-tuning process, these enzymes must have played an essential role in the progressive introduction of new amino acids, and in the refinement and standardization of the canonical nuclear genetic code observed in all extant organisms (functional convergent evolutionary hypothesis).