Role of post-transcriptional modifications of transfer RNA and post-translational modification of ribosomal protein in regulating translational accuracy in Saccharomyces cerevisiae

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UMBC Theses and Dissertations

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2017-01-01

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dissertations
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Biological Sciences

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Biological Sciences

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Abstract

Misreading errors are a frequent source of error during translation that result in incorporation of a wrong amino acid in the growing polypeptide chain and can potentially generate a misfolded protein. Studies have estimated misreading errors to occur with a frequency of 10-3 - 10-4 errors per codon. However, recent studies in E. coli have determined that the frequency of misreading errors is in fact 100-fold lower (10-6 errors per codon) for most near-cognate codons. I developed a novel reporter based assay that can be used to quantify errors by a single tRNA(Glu@UUC) in Saccharomyces cerevisiae. Using this reporter I find that the rate of misreading errors in yeast can vary from 10-6 - 10-4 errors per codon. I also find that the rate of errors in yeast is about 3-fold lower than that in bacteria. Another important conclusion made from this study is the occurrences of errors at the wobble position in yeast. Using the Beta-galactosidase reporter system developed in this study and the dual luciferase reporter system previously developed in our lab, I characterized the function of post-transcriptional modifications present in the anticodon stem loop of tRNA(Glu@UUC) and tRNA(Lys@UUU). I find that both mcm5s2U34 and t6A37 modification regulate decoding accuracy during translation in a codon context dependent manner. mcm5s2U34 was found to promote errors for A- ending codons, while restricting errors for G- ending codons. Similarly, t6A37 is found to restrict U�U mismatch at the first codon anticodon position but promotes the same error at the wobble position. Using genetic studies and our reporter system I established that Ctk1-dependent phosphorylation at serine 176 on ribosomal protein uS5 is critical in maintaining accuracy during translation. I show that loss of this phosphorylation site can increase misreading errors, while the presence of a phosphomimetic mutation at position 176 on uS5 can make a yeast strain hyper accurate. This study elucidates the important role played by modifications of the translational apparatus during decoding.