Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Biochemistry

Supervisor

Dr. David Haniford

Abstract

This thesis investigates the role that the RNA binding protein Hfq has in regulating the mobility of IS Elements and the expression of their transposase genes. Hfq is a known regulator of the IS Element IS10 and is a major component of sRNA pathways in E.coli. In this work, I demonstrate that through the use of a bioinformatics approach to identify a list of additional IS Elements from the IS Finder database, one can successfully identify IS Elements containing Hfq binding sites which may implicate Hfq regulation. One of the identified elements, IS1413, was characterized by transposition assays and transposase expression assays. These experiments demonstrated that the activity level of IS1413 decreases in the presence of Hfq. Another identified IS Element, IS10, had its 3’ end of its transposase mRNA transcript probed for the relevance of a putative Hfq binding site through mutational analysis, and through identifying that there are no post-transcriptional modifications at the 3’ end of the transcript. Although Hfq bound IS10 mRNA in vitro, genetic mutations to the putative Hfq binding site resulted in no difference in transposition activity. A growth condition was identified that increased IS10 transposition frequencies in wild type cells, but not in cells containing a rpoS gene disruption. Overall, these studies have identified another IS Element that changes activity in the presence of Hfq and have linked slow unaerated growth conditions to an increase in IS10 transposition frequencies in E.coli.


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Biochemistry Commons

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