Date of Award

2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

acs encodes a high affinity enzyme that permits survival during carbon starvation. As befits a survival gene, its transcription is subject to complex regulation. Previously, the Wolfe lab reported that CRP activates acs transcription by binding tandem DNA sites located upstream of the major acsP2 promoter and that the nucleoid protein IHF binds three specific sites located just upstream. The most proximal site (IHF III) exhibits reduced transcription compared to the full-length promoter or to a construct lacking all three IHF sites. The goal of my research was to understand how IHF III inhibits CRP-dependent acs transcription. First, I helped define the minimal system required for this IHF-dependent inhibition, showing it requires the promoter-distal CRP site and an amino acid residue located within a surface determinant of CRP that interacts with RNAP. Surprisingly, for a Class III promoter, disruption of this surface determinant caused significant changes in the activity and structure of both the full-length promoter and the construct with the single proximal IHF site. My collaborator, Dr. Bianca Sclavi (Laboratoire de Biotechnologies et Pharmacologie génétique Appliquée, Paris, France) showed that occupancy of IHF III mediates formation of a stalled unproductive transcription complex. This work was published in Molecular Microbiology.

I furthered this research, obtaining evidence that IHF III is actually a composite site consisting of two overlapping IHF sites that sit on opposing faces of the DNA helix. This composite appears to behave as a transcriptional regulatory switch. If IHF occupies one site, acs transcription occurs. If IHF occupies the opposing site, acs transcription is inhibited. Which site becomes occupied appears to involve occupancy of IHF II, which is located just upstream of IHF III.

This work demonstrates that the typical textbook view bacterial transcription is overly simplistic. In fact, bacterial transcription can be quite complex.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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