Farabaugh, Philip JCao, LingCao, Ling2019-10-112019-10-112017-01-0111765http://hdl.handle.net/11603/15043Misreading error frequency in mammalian cells during protein synthesis was reported to range from 10-2 to 10-5 error per codon. The studies reporting these error frequencies have measured several different errors by using various methods. To develop a more comprehensive understanding of translational accuracy in mammalian cells, I utilized a dual luciferase reporter system to quantify the frequency of all possible misreading events by tRNA_UUU^Lys in HEK293, HeLa, 22RV1 and NIH3T3 cell lines. The results showed that the pattern of misreading error frequency in these mammalian cell lines was similar but with distinct features. In addition, the difference in misreading error frequency at several error-prone codons by tRNA_UUU^Lys in mammalian cell lines did not vary as much as it did in E. coli and yeast. I speculate that roughly equivalent isoacceptor tRNA concentrations in mammalian cells led to this small variation in misreading frequency. In a second project, I developed a yeast Saccharomyces cerevisiae reporter system for errors by tRNA_UUG^Gln verifying that Gln 624 but not Gln 625 is an essential amino acid for ?-galactosidase that can be used to quantify misreading frequencies. Using this reporter system, I further tested the effects of tRNA modifications (mcm5 and s2 at U34, and ? at position 38) on translational fidelity. Loss of these tRNA modifications decreased misreading frequency at some error-prone codons, suggesting that tRNA modifications do not always benefit protein translation. I have observed errors involving novel base pair mismatches in both of my projects, broadening the set of all possible misreading events.This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.eduText