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 ...

Gcd10p andMet maturation. The chromatographic behavior of elongator and initiator tRNA Met on a RPC-5 column indicated that both species are altered structurally in gcd10⌬ cells, and analysis of base modifications revealed that 1-methyladenosine (m 1 A) is undetectable in gcd10⌬ tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNA Met , which also contains m 1 A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

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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 (m(1)G37), 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(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.