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Date of Award

Spring 5-15-2010

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The ribosome is a highly efficient machine, synthesizing polypeptides at a rate of ~20 amino acids per second. Multiple levels of regulation are required to maintain a high rate of protein synthesis at a low error frequency. The incorporation of amino acids by transfer RNAs (tRNAs) is a critical aspect of faithful translation. tRNAs have to be properly processed and modified to function in translation. Despite their small size, tRNAs contain extensive posttranscriptional nucleoside modifications. The formation ofN6-threonylcarbamoyladenosine (t6A) at nucleoside 37 in tRNAs decoding ANN (N= any base) is conserved across all domains of life. t6A is present next to the anticodon and in vitro work has demonstrated that the modified base stabilizes the codon:anticodon interaction to prevent frameshifting during the decoding process. However, there is a paucity of in vivo data to support the in vitro findings. Recently, Sua5 was described as a conserved protein required for the formation of t6A. To understand the functional role of Sua5 and the relevance of t6A in translation, we employed a variety of translational reporter assays. Our results conclude that cells depleted of Sua5 exhibit aberrant translation in all three phases: initiation, elongation and termination. These data provide in vivo evidence to support the role of Sua5 and t6A in maintaining translational fidelity. When we further investigated the role of Sua5 in tRNA homeostasis, we found that it exhibited synthetic sickness with a deletion inMAF1, a negative regulator of Pol III. This is consistent with the idea that increasing the pool of hypomodified tRNAs compounds the defects in codon misreading. Another consequence of Sua5-depletion is the loss of 40S ribosomes. To date, Xrn1 is the only exonuclease found to degrade mature 18S ribosomal RNA (rRNA). We found that Sua5 exhibits a genetic interaction with Xrn1. We provide preliminary data to show that Sua5 depletion slows down the decay of mutant rRNAs. Further analyses on the turnover of wild type and mutant rRNAs not engaged in translation in Sua5-depleted cells will shed light on the process of rRNA degradation.

Language

English (en)

Chair and Committee

Emily Cheng

Committee Members

Gregory D Longmore, Kendall J Blumer

Comments

Permanent URL: https://doi.org/10.7936/K7X928G2

Available for download on Thursday, May 15, 2110

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