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

• Author / Creator

  Payoe, Roshani

• 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.

• Subjects / Keywords

  • TRNA Identity
  • PppGpp
  • ACT domain
  • RelA
  • Stringent Response

• Graduation date

  Fall 2012

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3XC82

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.