ERA

Download the full-sized PDF of Molecular Studies on the RelA-Mediated (p)ppGpp Synthesis MechanismDownload the full-sized PDF

Analytics

Share

Permanent link (DOI): https://doi.org/10.7939/R3XC82

Download

Export to: EndNote  |  Zotero  |  Mendeley

Communities

This file is in the following communities:

Graduate Studies and Research, Faculty of

Collections

This file is in the following collections:

Theses and Dissertations

Molecular Studies on the RelA-Mediated (p)ppGpp Synthesis Mechanism Open Access

Descriptions

Other title
Subject/Keyword
tRNA Identity
pppGpp
ACT domain
RelA
Stringent Response
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Payoe, Roshani
Supervisor and department
Fahlman, Richard (Biochemistry)
Examining committee member and department
Wieden, Hans-Joachim (Biochemistry)
Owttrim, George (Biology)
Schultz, Michael C (Biochemistry)
MacMillan, Andrew (Biochemistry)
Department
Department of Biochemistry
Specialization

Date accepted
2012-09-28T06:50:52Z
Graduation date
2012-09
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
In Escherichia coli, the enzyme RelA catalyses the synthesis of (p)ppGpp in response to amino acid starvation. RelA activation requires the codon specific binding of a deacylated tRNA to the ribosomal A-site. By a poorly understood regulatory mechanism, RelA alternates between an inactive ribosome-bound and an active ribosome-free state. RelA activation on the ribosome terminates with the dissociation of the deacylated tRNA from the A-site. Inactivation of RelA off the ribosome is presumed to be via a conformational change in the C-terminal region of RelA We use an in vitro assay in combination with standard molecular mutagenesis techniques to gain further insight into two aspects of the RelA mediated (p)ppGpp synthesis mechanism: the first is the influence tRNA species has on the duration of (p)ppGpp synthesis. The second is the involvement of the ACT domain, a common regulatory domain of metabolic proteins, in the interaction with the ribosome and its function as a potential allosteric regulatory site in RelA. A tRNA can function as both a monitor of nutrient status in the cell and a co-activator of the enzyme RelA. E. coli contains 47 different tRNAs each with its own unique feature, one of which is the differences in A-site dissociation rates. We are the first to report that this idiosyncratic feature of the tRNA does indeed have an effect on the duration of (p)ppGpp synthesis in vitro. The C-terminal region of RelA is comprised of two domains with undefined function in RelA. In our characterisation of these domains, we not only identified a function to the ACT domain in RelA but also a novel mechanism of regulation where amino acid methionine is an allosteric inhibitor of RelA activity. Our molecular studies into the RelA mediated (p)ppGpp synthesis addresses two aspect of this mechanism that until now has been left unexplored, and thus has significantly contributed to our knowledge of this potent survival response in bacteria.
Language
English
DOI
doi:10.7939/R3XC82
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Payoe R and Fahlman RP (2011) Dependence of RelA-Mediated (p)ppGpp Formation on tRNA Identity. Biochemistry, 50(15): 3075-3083

File Details

Date Uploaded
Date Modified
2014-04-30T23:44:19.855+00:00
Audit Status
Audits have not yet been run on this file.
Characterization
File format: pdf (Portable Document Format)
Mime type: application/pdf
File size: 9711660
Last modified: 2015:10:18 01:35:52-06:00
Filename: Payoe_Roshani_Fall 2012.pdf
Original checksum: 881ebb4396a896bda495224f1032a7b6
Well formed: true
Valid: true
Page count: 173
Activity of users you follow
User Activity Date