All ETDs from UAB

Advisory Committee Chair

David M Bedwell

Advisory Committee Members

Ching-Yi Chen

James F Collawn

Kim M Keeling

Sunnie R Thompson

Mark Walter

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Translation termination is an essential eukaryotic process that facilitates the end of protein synthesis and mediates the release of the nascent peptide chain. Two release factors, eRF1 and eRF3, with the help of a ribosome recycling factor assist in this process. eRF1 and eRF3 form a stable complex through conserved regions in their C-terminal domains. The eRF1/eRF3 complex, along with GTP, is absolutely vital for efficient termination in vitro and in vivo. The role of eRF1 during translation termination is to recognize the stop codon located in the ribosome aminoacyl site, stimulate GTP hydrolysis of eRF3 and enable peptide release through its GGQ motif. eRF3 aids this process in a GTP-dependent manner and through promoting conformational changes in eRF1. Although recent advancements have elucidated how prokaryotic release factors directly recognize each stop codon (UAA, UAG and UGA), this has not aided our understanding of the mechanism of stop codon recognition utilized by eRF1, since eRF1 3-dimensitional structure is significantly different. To gain a better understanding of how eRF1 is able to mediate this essential function, we extensively analyzed two motifs known to play a significant role in stop codon recognition. We found that the YCF motif most often contributed to eRF1 selective stop codon specificity while changes in the TASNIKS motif displayed non-specific stop codon selectivity. Through our studies we also found that the abundance of eRF1 protein and eRF1 mRNA (SUP45 mRNA) is regulated by a post-transcriptional regulatory mechanism. The major component for this increase was contributed by an increase in SUP45 mRNA stability. We show using in vitro and in vivo studies that limiting functional termination complexes result in increased levels of SUP45 mRNA and eRF1 protein.

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