Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Regulatory Biology

Department

College of Sciences and Health Professions

First Advisor

Komar, Anton

Subject Headings

Molecular Biology

Abstract

The process of translation in all living cells is performed by ribosomes and is divided into four major steps (initiation, elongation, termination and ribosome recycling). Ribosomes consist of ribosomal RNA (rRNA) and proteins and are composed of two subunits small and large. There are three major sites for tRNA binding within ribosome: the (aminoacyl) A-site which accepts the aminoacyl (aa)-tRNA; the P-site, where the peptidyl-tRNA is formed and the (exit) E-site, where deacylated tRNA exits the ribosome. These sites are formed by both rRNA and ribosomal proteins. Though rRNA are involved in the catalysis of protein synthesis, the contribution of individual ribosomal proteins to protein synthesis is not fully understood in molecular terms. Yeast ribosomal protein uS9/yRps16, is universally conserved and is located on the solvent side of the small ribosomal subunit. It has a long protruding C-terminal tail (CTT) which extends towards the mRNA cleft. This CTT contributes to the formation of the ribosomal P-site. uS9/yRps16 last positively charged C terminal residue (Arg), is invariably conserved and is believed to enhance interaction of the negatively charged initiator tRNA, when base-paired to AUG codon in the P site. However, biochemical evidence in support of this notion is limited. Our biochemical analysis of the uS9 mutants showed that the C terminus of uS9 plays an important role during translation initiation. We found that uS9 C-terminal residues (their exact location and nature) are critical for efficient recruitment of the eIF2•GTP•MettRNAiMet ternary complex and for responding properly to an AUG codon in the P-site, during scanning phase of initiation. These residues also regulate hydrolysis of GTP (from eIF2•GTP•Met-tRNAiMet complex) to GDP and Pi. Furthermore, deletion of the last two residues of uS9 CTT, exhibits resistance to anisomycin, decreased association of elongation factor eEF1A to polyribosomes at the A-site and decreased programmed ribosomal frameshift (PRF) efficiency, thus showing that uS9 C terminal region modulates elongation fidelity. Therefore, we propose that uS9 CTT is critical for proper control of the complex interplay of events surrounding the accommodation of initiator and elongator tRNAs in the P- and A-sites of the ribosome.

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