Glenn R. Björk

Improvement of reading frame maintenance is a common function for several tRNA modifications

Transfer RNAs from all organisms contain many modified nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their influence on different reactions in which tRNA participates suggest that each modified nucleoside may have its own specific function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modification was higher compared with the corresponding wild‐type controls, these modifications have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl‐tRNA slippage, which is influenced by the hypomodified tRNA in two ways: (i) a hypomodified tRNA in the ternary complex may decrease the rate by which the complex is recruited to the A‐site and thereby increasing peptidyl‐tRNA slippage; or (ii) a hypomodified peptidyl‐tRNA may be more prone to slip than its fully modified counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modified nucleosides.

A primordial tRNA modification required for the evolution of life

The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present‐day organisms. We show that one such feature may be the modified nucleoside 1‐methylguanosine (m1G37), which prevents frameshifting and is present adjacent to and 3′ of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m1G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m1G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m1G37)methyltransferase also synthesizes 1‐methylinosine and participates in the formation of the Y‐base (yW). Our results suggest that m1G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m1G37)methyltrans ferase is part of the minimal set of gene products required for life.

Eukaryotic Wobble Uridine Modifications Promote a Functionally Redundant Decoding System

ABSTRACT The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm5) or a 5-carbamoylmethyl (ncm5) side chain and sometimes an additional 2-thio (s2) or 2′-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5U34- and some ncm5U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

How translational accuracy influences reading frame maintenance

Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from a second error occurring after recruitment of an errant aminoacyl‐tRNA, either spontaneous dissociation causing premature termination or translational frameshifting. This is clearest in programmed translational frameshifting where the mRNA programs errant reading by a near‐cognate tRNA; this error promotes a second frameshifting error (a dual‐error model of frameshifting). The same mechanism can explain frameshifting by suppressor tRNAs, even those with expanded anticodon loops. The previous model that suppressor tRNAs induce quadruplet translocation now appears incorrect for most, and perhaps for all of them. We suggest that the ‘spontaneous’ tRNA‐induced frameshifting and ‘programmed’ mRNA‐induced frameshifting use the same mechanism, although the frequency of frameshifting is very different. This new model of frameshifting suggests that the tRNA is not acting as the yardstick to measure out the length of the translocation step. Rather, the translocation of 3 nucleotides may be an inherent feature of the ribosome.

Transfer RNA modification, temperature and DNA superhelicity have a common target in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene

Full expression of the virulence genes of Shigella flexneri requires the presence of two modified nucleosides in the tRNA [queuosine, Q34, present in the wobble position (position 34) and 2‐methylthio‐N6‐isopentenyladenosine (ms2i6A37, adjacent to and 3′ of the anticodon)]. The synthesis of these two nucleosides depends on the products of the tgt and miaA genes respectively. We have shown that the intracellular concentration of the virulence‐related transcriptional regulator VirF is reduced in the absence of either of these modified nucleosides. The intracellular concentration of VirF is correlated with the expression of the virulence genes. Overproduction of VirF in the tgt and the miaA mutants suppressed the less virulent (tgt) or the avirulent (miaA) phenotypes respectively, caused by the tRNA modification deficiency. This suggests that the primary result of undermodification of the tRNA is a poor translation of virF mRNA and not of any other mRNA whose product acts downstream of the action of VirF. Shigella showed no virulence phenotypes at 30°C, but forced synthesis of VirF at 30°C induced the virulence phenotype at this low temperature. In addition, removal of the known gene silencer H‐NS by a mutation in its structural gene hns increased the synthesis of VirF at low temperature and thus induced a virulent phenotype at 30°C. Conversely, decreased expression of VirF at 37°C induced by the addition of novobiocin, a known inhibitor of gyrase, led to an avirulent phenotype. We conclude that tRNA modification, temperature and superhelicity have the same target – the expression of VirF – to influence the expression of the central regulatory gene virB and thereby the virulence of Shigella. These results further strengthen the suggestion that the concentration of VirF is the critical factor in the regulation of virulence in Shigella. In addition, they emphasize the role of the bacterial translational machinery in the regulation of the expression of virulence genes which appears here quantitatively as important as the well‐established regulation on the transcriptional level.

TRANSFER RNA MODIFICATION: INFLUENCE ON TRANSLATIONAL FRAMESHIFTING AND METABOLISM

Transfer RNA modification improves the rate of aa‐tRNA selection at the A‐site and the fitness in the P‐site and thereby prevents frameshifting according to a new model how frameshifting occurs [Qian et al. (1998) Mol. Cell 1, 471–482]. Evidence that the presence of various modified nucleosides in tRNA also influences central metabolism, thiamine metabolism, and bacterial virulence is reviewed.

A Gripping Tale of Ribosomal Frameshifting: Extragenic Suppressors of Frameshift Mutations Spotlight P-Site Realignment

SUMMARY Mutants of translation components which compensate for both −1 and +1 frameshift mutations showed the first evidence for framing malleability. Those compensatory mutants isolated in bacteria and yeast with altered tRNA or protein factors are reviewed here and are considered to primarily cause altered P-site realignment and not altered translocation. Though the first sequenced tRNA mutant which suppressed a +1 frameshift mutation had an extra base in its anticodon loop and led to a textbook “yardstick” model in which the number of anticodon bases determines codon size, this model has long been discounted, although not by all. Accordingly, the reviewed data suggest that reading frame maintenance and translocation are two distinct features of the ribosome. None of the −1 tRNA suppressors have anticodon loops with fewer than the standard seven nucleotides. Many of the tRNA mutants potentially affect tRNA bending and/or stability and can be used for functional assays, and one has the conserved C74 of the 3′ CCA substituted. The effect of tRNA modification deficiencies on framing has been particularly informative. The properties of some mutants suggest the use of alternative tRNA anticodon loop stack conformations by individual tRNAs in one translation cycle. The mutant proteins range from defective release factors with delayed decoding of A-site stop codons facilitating P-site frameshifting to altered EF-Tu/EF1α to mutant ribosomal large- and small-subunit proteins L9 and S9. Their study is revealing how mRNA slippage is restrained except where it is programmed to occur and be utilized.

Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli

In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an α2β2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent.

Isolation of mutants of Escherichia coli lacking 5-methyluracil in transfer ribonucleic acid or 1-methylguanine in ribosomal RNA

Abstract A screening procedure for mutants of Escherichia coli with RNA possessing methyl-group acceptor ability in vitro was developed. Ten such mutants were found and characterized. RNA from six of them accepted methyl groups in vitro only into transfer RNA, with the exclusive formation of 5-methyluracil, when enzyme extract from the parent strain was used. Analysis of the content of methylated compounds formed in vivo showed a normal distribution of methylated constituents in both ribosomal RNA and tRNA except for a partial or complete lack of 5-methyluracil in tRNA. RNA from three other mutants accepted methyl groups in vitro only into rRNA, when methylated, with the formation of N2-methylguanine and some unidentified compound(s). However, these three mutants appeared to have a normal content of methylated compounds in both rRNA and tRNA when analyzed in vivo. RNA from another mutant accepted methyl groups in vitro into tRNA, with the formation of 5-methyluracil, and into rRNA with the formation of 1-methylguanine and to a lesser extent 5-methyluracil. In vivo this mutant was shown to lack 5-methyluracil in its tRNA and 1-methylguanine in its rRNA. From genetic data it was concluded that these deficiencies were due to two independent mutations. The finding and characterization of the mutants described show that the complete absence of 5-methyluracil and tRNA and 1-methylguanine in rRNA is not lethal. The existence of the mutants suggests that different enzymes are responsible for the formation of 5-methyluracil in rRNA and tRNA respectively. The same conclusion is probably justified also for the modification of guanine to 1-methylguanine.

A New Model for Phenotypic Suppression of Frameshift Mutations by Mutant tRNAs

According to the prevailing model, frameshift-suppressing tRNAs with an extra nucleotide in the anticodon loop suppress +1 frameshift mutations by recognizing a four-base codon and promoting quadruplet translocation. We present three sets of experiments that suggest a general alternative to this model. First, base modification should actually block such a four-base interaction by two classical frameshift suppressors. Second, for one Salmonella suppressor tRNA, it is not mutant tRNA but a structurally normal near cognate that causes the +1 shift in-frame. Finally, frameshifting occurs in competition with normal decoding of the next in-frame codon, consistent with an event that occurs in the ribosomal P site after the translocation step. These results suggest an alternative model involving peptidyl-tRNA slippage at the classical CCC-N and GGG-N frameshift suppression sites.