A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

Abstract

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis of possible errors. We recently used a highly quantitative system to measure the frequency of all types of misreading errors by a single tRNA in E. coli. That study found a wide variation in error frequencies among codons; a major factor causing that variation is competition between the correct (cognate) and incorrect (near-cognate) aminoacyl-tRNAs for the mutant codon. Here we extend that analysis to measure the frequency of missense errors by two tRNAs in a eukaryote, the yeast Saccharomyces cerevisiae. The data show that in yeast errors vary by codon from a low of 4 3 10 À5 to a high of 6.9 3 10 À4 per codon and that error frequency is in general about threefold lower than in E. coli, which may suggest that yeast has additional mechanisms that reduce missense errors. Error rate again is strongly influenced by tRNA competition. Surprisingly, missense errors involving wobble position mispairing were much less frequent in S. cerevisiae than in E. coli. Furthermore, the error-inducing aminoglycoside antibiotic, paromomycin, which stimulates errors on all error-prone codons in E. coli, has a more codon-specific effect in yeast.

Distinct response of yeast ribosomes to a miscoding event during translation

“…) (Salas-Marco and Bedwell 2005;Kramer et al 2010), the acceptance of aminoacyl-tRNAs and eRF1 d eRF3 on near-cognate codons was minimal in the in vitro system. Moreover, as in bacteria, the aminoglycoside class of antibiotics stimulated misreading during tRNA selection and inhibited release factor function.…”

“…Similarly, paromomycin appears to stimulate miscoding in S. cerevisiae as determined with a series of reporter constructs Eyler and Green (Fan-Minogue and Bedwell 2008;Kramer et al 2010). Here, we evaluated the effects of paromomycin on tRNA and eRF selection in our in vitro reconstituted system.…”

“…This evolved level of fidelity during translation is not the same in all organisms; indeed, more complex organisms appear to have greater overall fidelity than simpler ones. For example, the in vivo miscoding frequencies in yeast appear to be between 1 3 10 À4 and 5 3 10 À4 misreading events per codon (Stansfield et al 1998;Rakwalska and Rospert 2004;Salas-Marco and Bedwell 2005;Plant et al 2007;Kramer et al 2010), about 10-fold less frequent than the miscoding frequencies measured in vivo in Escherichia coli (Parker 1989;Kramer and Farabaugh 2007). In higher eukaryotes errors may be even less frequent (Martin et al 1989).…”

The yeast rapid tRNA decay pathway competes with elongation factor 1A for substrate tRNAs and acts on tRNAs lacking one or more of several modifications

“…Third, to ensure high-accuracy translation (Zaher and Green 2009;Kramer et al 2010), all tRNAs require features to uniquely decode their cognate codon, including an extensive set of anticodon loop modifications for highly specific codon:anticodon pairing Agris et al 2007), and other sequence elements (Cochella and Green 2005;Ledoux et al 2009). Fourth, all tRNAs are required to be rugged enough to survive constant use in translation but flexible enough to withstand the bending and contortions that occur during ribosome transit Yarus et al 2003;Schmeing et al 2009).…”

Different types of secondary information in the genetic code

“…The efficiency at which a codon is translated is determined by the amount of cognate tRNA activity available, which is in turn determined to a large extent by the tRNA gene copy number content (Gouy and Gautier 1982;Ikemura 1985;Dong et al 1996;Berg and Kurland 1997;Percudani et al 1997;Duret 2000;Kanaya et al 2001;Tuller et al 2010;Novoa et al 2012), whereas fidelity is impacted by competition with noncognate tRNAs (Kramer and Farabaugh 2007;Kramer et al 2010;Reynolds et al 2010;Lamichhane et al 2013a). If all codons were equally distributed among all mRNAs, all tRNAs were present and equally active at equimolar abundance, and high fidelity prevailed, there would be little if any consequence of synonymous or rare codon use, except on mRNA structure, stability, and other features such as splicing and transcription factor-binding sites (Chamary and Hurst 2005;Chamary et al 2006, and references therein; Parmley et al 2007;Gu et al 2010;Goodman et al 2013;Stergachis et al 2013).…”