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Date Permissions Signed

7-2017

Date of Award

Summer 2017

Document Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Spiegel, P. Clint

Second Advisor

Prody, Gerry

Third Advisor

Antos, John M.

Abstract

Ribosomes are the macromolecular machines responsible for protein synthesis across all domains of life. Translation of genetic information into a polypeptide by ribosomes is facilitated by a multitude of proteins called translation factors, many of which belong to the guanosine 5’ triphosphate hydrolase (GTPase) superfamily that utilize the hydrolysis of GTP to exert their function. Many naturally occurring antibiotics inhibit protein biosynthesis by targeting the bacterial ribosome or associated translation factors and with increasing antibiotic resistance due to bacterial evolution, the importance of studying ribosome-translation factor interactions is amplified. Determination of high-resolution structures of the ribosome has significantly bolstered our understanding of translation, yet crucial mechanisms remain poorly understood, including the mechanism of ribosome-dependent GTPase binding and activation. Several GTPases harbor conserved G domains, which bind to conserved regions of the ribosomal subunit interface. It has been observed that a conserved sequence in the C-terminal domain (CTD) of ribosomal protein L12 makes direct contacts with the G’ subdomain of the GTPase elongation factor G (EF-G), and L12-depletion studies have demonstrated that the L12 protein is required for binding and GTP hydrolysis by EF-G on the ribosome.

This work aims to identify key residues in the conserved binding region of L12 that are critical for GTPase recruitment and activation. Based on careful structural study of the L12-GTPase binding interface, single amino acid point mutations (L12-K66A, -K66D, -K82A, -K82D, -K85A, -K85D, -T77W, and -T77A) were generated by site-directed mutagenesis to assess functionality of conserved residues. By an established L12 depletion protocol, wild-type (wt) L12 was completely removed, followed by reconstitution of L12 mutant proteins to ribosomal complexes for analysis of their ability to stimulate GTPase activity.

Here, we show that removal of the G’ subdomain reduces activity of EF-G by 60% in both the presence and absence of L12 on the ribosome while wt EF-G activity is completely abrogated in the presence of L12-depleted ribosomes. Furthermore, reconstitution of L12-depleted ribosomes with an L12 CTD mutant was insufficient for restoration of any lost activity of wt EF-G, indicating that the L12 N-terminal domain is required for proper function of L12. Finally, substitution of an aspartic acid in place of the highly conserved lysine 82 in the CTD of L12 decreased wt EF-G activity to 20%, marking the importance of charge-charge interactions at the L12-GTPase binding interface. Together, these results support and extend understanding of the essential role of L12 in recruitment and activation of ribosome-dependent GTPases, paving the way for future work on development of new antibiotic molecules targeting these interactions.

Type

Text

Publisher

Western Washington University

OCLC Number

1000154284

Digital Format

application/pdf

Genre/Form

Academic theses

Language

English

Rights

Copying of this thesis in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this thesis for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.

Included in

Chemistry Commons

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