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.     

Decoding the genome: a modified view

Transfer RNA’s role in decoding the genome is critical to the accuracy and efficiency of protein synthesis. Though modified nucleosides were identified in RNA 50 years ago, only recently has their importance to tRNA’s ability to decode cognate and wobble codons become apparent. RNA modifications are ubiquitous. To date, some 100 different posttranslational modifications have been identified. Modifications of tRNA are the most extensively investigated; however, many other RNAs have modified nucleosides. The modifications that occur at the first, or wobble position, of tRNA’s anticodon and those 3′-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modifications respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (e.g. AAA/G for lysine versus AAU/C asparagine). In contrast, other modifications expand wobble codon recognition, such as U·U base pairing, for tRNAs that respond to multiple codons of a 4-fold degenerate codon box (e.g. GUU/A/C/G for valine). Whether restricting codon recognition, expanding wobble, enabling translocation, or maintaining the messenger RNA, reading frame modifications appear to reduce anticodon loop dynamics to that accepted by the ribosome. Therefore, we suggest that anticodon stem and loop domain nucleoside modifications allow a limited number of tRNAs to accurately and efficiently decode the 61 amino acid codons by selectively restricting some anticodon–codon interactions and expanding others.     

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.     

Wobble position modified nucleosides evolved to select transfer RNA codon recognition: a modified-wobble hypothesis.

While recognized that some wobble exists in the base pairing of the first base of the tRNA anticodon with the third of the codon, specific base modifications have evolved to select particular codons. This modified-wobble theory would be exemplified by a single codon recognition imposed on the anticodon by modification of the tRNA wobble position nucleoside.     

The effect of specific structural modification on the biological activity of E. coli arginine tRNA.

Escherichia coli arginine tRNA1 has been modified at position s2C32 with iodoacetamide and a spin labelled derivative. The small effects on the charging ability of tRNA by the modifiications suggest that the synthetase does not bind to the tRNA in this region of the anticodon loop before the anticodon. A ternary complex of elongation factor Tu, GTP and the modified Arg-tRNA, can be formed allowing future studies of enzymatic binding to the ribosome. Using the triplet binding assay the native Arg-tRNA1 decodes all 4 codons beginning with CG. The modified Arg-tRNA1 has a restricted decoding but the decoding pattern is still unusual according to the Wobble Hypothesis.     

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.     

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.     

Role of modified nucleosides of yeast tRNAPhe in ribosomal binding

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.     

Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.     

Familial dysautonomia (FD) patients have reduced levels of the modified wobble nucleoside mcmsU in tRNA

Familial dysautonomia (FD) is a recessive neurodegenerative genetic disease. FD is caused by a mutation in the IKBKAP gene resulting in a splicing defect and reduced levels of full length IKAP protein. IKAP homologues can be found in all eukaryotes and are part of a conserved six subunit protein complex, Elongator complex. Inactivation of any Elongator subunit gene in multicellular organisms cause a wide range of phenotypes, suggesting that Elongator has a pivotal role in several cellular processes. In yeast, there is convincing evidence that the main role of Elongator complex is in formation of modified wobble uridine nucleosides in tRNA and that their absence will influence translational efficiency. To date, no study has explored the possibility that FD patients display defects in formation of modified wobble uridine nucleosides as a consequence of reduced IKAP levels. In this study, we show that brain tissue and fibroblast cell lines from FD patients have reduced levels of the wobble uridine nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U). Our findings indicate that FD could be caused by inefficient translation due to lower levels of wobble uridine nucleosides.     

The RNA acetyltransferase driven by ATP hydrolysis synthesizes N4-acetylcytidine of tRNA anticodon

The wobble base of Escherichia coli elongator tRNA(Met) is modified to N(4)-acetylcytidine (ac(4)C), which is thought to ensure the precise recognition of the AUG codon by preventing misreading of near-cognate AUA codon. By employing genome-wide screen of uncharacterized genes in Escherichia coli ('ribonucleome analysis'), we found the ypfI gene, which we named tmcA (tRNA(Met) cytidine acetyltransferase), to be responsible for ac(4)C formation. TmcA is an enzyme that contains a Walker-type ATPase domain in its N-terminal region and an N-acetyltransferase domain in its C-terminal region. Recombinant TmcA specifically acetylated the wobble base of E. coli elongator tRNA(Met) by utilizing acetyl-coenzyme A (CoA) and ATP (or GTP). ATP/GTP hydrolysis by TmcA is stimulated in the presence of acetyl-CoA and tRNA(Met). A mutation study revealed that E. coli TmcA strictly discriminates elongator tRNA(Met) from the structurally similar tRNA(Ile) by mainly recognizing the C27-G43 pair in the anticodon stem. Our findings reveal an elaborate mechanism embedded in tRNA(Met) and tRNA(Ile) for the accurate decoding of AUA/AUG codons on the basis of the recognition of wobble bases by the respective RNA-modifying enzymes.     

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.     

A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine

Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA.