Transfer RNAs from all organisms contain many modi®ed nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their in¯uence on different reactions in which tRNA participates suggest that each modi®ed nucleoside may have its own speci®c function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modi®cation was higher compared with the corresponding wild-type controls, these modi®cations have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl-tRNA slippage, which is in¯uenced by the hypomodi®ed tRNA in two ways: (i) a hypomodi®ed 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 hypomodi®ed peptidyl-tRNA may be more prone to slip than its fully modi®ed counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modi®ed nucleosides. Keywords: frameshift/modi®ed nucleoside/tRNA/ translation IntroductionThe capacity of the translation apparatus has evolved to read long messages and thereby to make sophisticated proteins required for life as we see it today. Although the translation apparatus has the ability to decode faithfully, errors occur with frequencies of 10 ±3 ±10 ±4 per codon (Kurland et al., 1996). Most missense errors are not harmful to proteins, since many amino acids can be substituted without affecting the stability or the activity of the protein. In sharp contrast to the missense errors, almost all of the frameshift errors are detrimental to the synthesis of a functional protein, since following such a shift in frame the amino acid sequence becomes completely different and eventually the ribosome usually encounters a stop codon. This results in a truncated, usually unstable or inactive, peptide. Clearly, during evolution, features of the translation apparatus that are pivotal for reading frame maintenance have evolved. To understand the mechanism by which the ribosome traverses the mRNA in a faithful manner, one has to unravel the features of the translation apparatus that are important for maintaining the correct reading frame.There are several examples of how frameshift errors occur by peptidyl-tRNA slippage induced by a pause occurring in the A-site (reviewed in Farabaugh, 1997;Farabaugh and Bjo Èrk, 1999; see also Figure 1). The length of the pause in the A-site, the ®tness of the tRNA in the P-site and the mRNA sequence determine the frequency of slippage by the peptidyl-tRNA and thus the frequency of frameshift. Changes in the tRNA structure, such as that induced by a de®ciency of a modi®ed nucleoside, may therefore affect reading frame maintenance. Modi®ed nucleosides are derivatives of the four major nucleosides U, C, A and G, and at present 81 different modi®ed nucleosides have ...

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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.

Transfer RNA (tRNA) from all organisms on this planet contains modified nucleosides, which are derivatives of the four major nucleosides. tRNA from Escherichia coli/Salmonella enterica serovar Typhimurium contains 33 different modified nucleosides, which are all, except one (Queuosine [Q]), synthesized on an oligonucleotide precursor, which by specific enzymes later matures into tRNA. The structural genes for these enzymes are found in mono- and polycistronic operons, the latter of which have a complex transcription and translation pattern. The synthesis of the tRNA-modifying enzymes is not regulated similarly, and it is not coordinated to that of their substrate, the tRNA. The synthesis of some of them (e.g., several methylated derivatives) is catalyzed by one enzyme, which is position and base specific, whereas synthesis of some has a very complex biosynthetic pathway involving several enzymes (e.g., 2-thiouridines, N  6-cyclicthreonyladenosine [ct6A], and Q). Several of the modified nucleosides are essential for viability (e.g., lysidin, ct6A, 1-methylguanosine), whereas the deficiency of others induces severe growth defects. However, some have no or only a small effect on growth at laboratory conditions. Modified nucleosides that are present in the anticodon loop or stem have a fundamental influence on the efficiency of charging the tRNA, reading cognate codons, and preventing missense and frameshift errors. Those that are present in the body of the tRNA primarily have a stabilizing effect on the tRNA. Thus, the ubiquitous presence of these modified nucleosides plays a pivotal role in the function of the tRNA by their influence on the stability and activity of the tRNA.

Transfer RNA modification improves the rate of aatRNA 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)

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Eukaryotic ribosomal frameshift signals generally contain two elements, a heptanucleotide slippery sequence (XXXYYYN) and an RNA secondary structure, often an RNA pseudoknot, located downstream. Frameshifting takes place at the slippery sequence by simultaneous slippage of two ribosome-bound tRNAs. All of the tRNAs that are predicted to decode frameshift sites in the ribosomal A-site (XXXYYYN) possess a hypermodified base in the anticodon-loop and it is conceivable that these modifications play a role in the frameshift process. To test this, we expressed slippery sequence variants of the coronavirus IBV frameshift signal in strains of Escherichia coli unable to modify fully either tRNA(Lys) or tRNA(Asn). At the slippery sequences UUUAAAC and UUUAAAU (underlined codon decoded by tRNA(Asn), anticodon 5' QUU 3'), frameshifting was very inefficient (2 to 3%) and in strains deficient in the biosynthesis of Q base, was increased (AAU) or decreased (AAC) only two-fold. In E. coli, therefore, hypomodification of tRNA(Asn) had little effect on frameshifting. The situation with the efficient slippery sequences UUUAAAA (15%) and UUUAAAG (40%) (underlined codon decoded by tRNA(Lys), anticodon 5' mnm5s2UUU 3') was more complex, since the wobble base of tRNA(Lys) is modified at two positions. Of four available mutants, only trmE (s2UUU) had a marked influence on frameshifting, increasing the efficiency of the process at the slippery sequence UUUAAAA. No effect on frameshifting was seen in trmC1 (cmnm5s2UUU) or trmC2 (nm5s2UUU) strains and only a very small reduction (at UUUAAAG) was observed in an asuE (mnm5UUU) strain. The slipperiness of tRNA(Lys), therefore, cannot be ascribed to a single modification site on the base. However, the data support a role for the amino group of the mnm5 substitution in shaping the anticodon structure. Whether these conclusions can be extended to eukaryotic translation systems is uncertain. Although E. coli ribosomes changed frame at the IBV signal (UUUAAAG) with an efficiency similar to that measured in reticulocyte lysates (40%), there were important qualitative differences. Frameshifting of prokaryotic ribosomes was pseudoknot-independent (although secondary structure dependent) and appeared to require slippage of only a single tRNA.