Codon-specific effects of tRNA anticodon loop modifications on translational misreading errors in the yeastSaccharomyces cerevisiae

Abstract

Protein synthesis requires both high speed and accuracy to ensure a healthy cellular environment. Estimates of errors during protein synthesis in Saccharomyces cerevisiae have varied from 10−3 to 10−4 errors per codon. Here, we show that errors made by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\rm{tRNA}}^{\rm Glu}_{\rm UUC}$\end{document} in yeast can vary 100-fold, from 10−6 to 10−4 errors per codon. The most frequent errors require a G•U mismatch at the second position for the near cognate codon GGA (Gly). We also show, contrary to our previous results, that yeast tRNAs can make errors involving mismatches at the wobble position but with low efficiency. We have also assessed the effect on misreading frequency of post-transcriptional modifications of tRNAs, which are known to regulate cognate codon decoding in yeast. We tested the roles of mcm5s2U34 and t6A37 and show that their effects depend on details of the codon anticodon interaction including the position of the modification with respect to the base mismatch and the nature of that mismatch. Both mcm5 and s2 modification of wobble uridine strongly stabilizes G2•U35 mismatches when \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\rm{tRNA}}^{\rm Glu}_{\rm UUC}$\end{document} misreads the GGA Gly codon but has weaker effects on other mismatches. By contrast, t6A37 destabilizes U1•U36 mismatches when \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\rm{tRNA}}^{\rm Lys}_{\rm UUU}$\end{document} misreads UAA or UAG but stabilizes mismatches at the second and wobble positions.

The problem of genetic code misreading during protein synthesis

“…Mitigating such errors requires a better understanding of their causes. Available data on misreading reveal that errors are skewed toward events involving a few specific base mismatches (Blanchet et al, ; Joshi et al, ; Manickam, Nag, Abbasi, Patel, & Farabaugh, ; Z. Zhang, Shah, & Bondarenko, ). The most efficient misreading events involve one of three base mismatches: G•U, U•U, and C•U (bases shown in codon•anticodon order; Figure ).…”

“…The most efficient misreading events involve one of three base mismatches: G•U, U•U, and C•U (bases shown in codon•anticodon order; Figure ). Frequent errors have been observed for all tRNAs with a middle position U 35 in the anticodon mismatching with a middle position G 2 in the codon (G 2 •U 35 mismatches) in E. coli (Manickam et al, ) and in S. cerevisiae (Joshi et al, ). G 2 •U 35 mismatch errors were independently observed by mass spectrometry in E. coli and mouse CHO cells (Z. Zhang et al, ).…”

“…Importantly, almost all other mismatches induce errors with only very low efficiency, no more than about 2 × 10 −6 per codon, whereas errors involving the three mismatches described above are much more frequent, up to 3.6 × 10 −3 per codon in bacteria (Kramer & Farabaugh, ; Manickam et al, ) and 7 × 10 −4 per codon in yeast (Joshi et al, ; Kramer et al, ). The 100 to 1,000‐fold difference in apparent error frequency may underestimate the range of errors because the low activity recorded for most codons results from apparent enzyme activities near the background of the assay.…”

“…First, errors are significantly more frequent overall in E. coli than in S. cerevisiae . Direct comparisons of error frequencies for (Kramer et al, ; Kramer & Farabaugh, ) and (Joshi et al, ; Manickam, Joshi, Bhatt, & Farabaugh, ) show that errors in E. coli average about threefold to fivefold greater than in S. cerevisiae . Because the frequency of errors is sensitive to the abundance of the cognate tRNA that competes for the misread codon, this difference could reflect a difference in the abundances of these competing cognate tRNAs or, for misreading of nonsense codons, differences in the availability of peptide release factors.…”

“…However, their effect on misreading is more complicated. In bacteria, the presence of mnm 5 modification strongly decreases wobble errors by but equally strongly increases errors by (Manickam et al, ), whereas the presence of mcm 5 in S. cerevisiae tends to change accuracy by a smaller ratio and generally increases misreading (Joshi et al, ). Importantly, with equivalently modified tRNAs carrying the same s 2 U 34 wobble modification, the error frequency in S. cerevisiae is still much less than in E. coli , suggesting that the difference is not a result of wobble modification.…”