The Escherichia coligal operon: Cross-talk between positive and negative regulation of transcription

Date

1995

Authors

Dalma-Weiszhausz, Dennise Deborah

Journal Title

Journal ISSN

Volume Title

Publisher

ProQuest Dissertations & Theses

YU Faculty Profile

Abstract

The regulation of gene expression is pivotal for cellular survival. The repression and activation of transcriptional regulation require a complex interplay of protein-DNA and protein-protein interactions. The aim of this thesis is to quantitatively and structurally describe the interactions that occur between DNA-bound proteins that regulate transcription, the DNA distortions brought about by them, and the putative role of DNA conformation on the regulation of transcriptional initiation. The Escherichia coli gal operon was chosen for this study since regulation of this operon is conferred by a small, but well characterized number of proteins, namely, GalR and the cAMP receptor protein (CAP).;The existence of a macromolecular repression complex that potentially includes Gal repressor (GalR), RNAP polymerase (RNAP) and the cAMP-CAP complex was investigated. This complex would insure that upon activation, RNAP would be poised to immediately start the process of transcriptional initiation. For this purpose, a thermodynamic analysis of the interactions that occur upon the binding of repressor, activator and RNAP was performed using quantitative DNase I footprinting. This technique allows the partitioning of the binding free energy into the intrinsic (protein-DNA) and the cooperative (protein-protein) energies of the protein-DNA association reactions.;These studies yielded the following results: (1) RNAP and GalR bind to DNA independently, arguing against a hypothesis that RNAP might be uniquely responsible in aiding GalR in the formation of a macromolecular nucleoprotein complex; (2) CAP and RNAP bind to adjacent regions in the gal promoter DNA in a highly cooperative fashion and alter the DNA structure, as evidenced by an altered sensitivity to enzymatic and chemical nucleases. Among the sites displaying an altered sensitivity, one is of particular interest, since it falls exactly within the {dollar}O\sb{lcub}I{rcub}{dollar} sequence; (3) the binding of LacI or GalR to the {dollar}O\sb{lcub}E{rcub}{dollar} site, just upstream of the CAP binding site, modulates the CAP-RNAP cooperativity, which might be a part of the mechanism of repression.;These studies define a hierarchy of the interactions that occur within a transcription pre-initiation complex. The communication between the mechanisms of activation and repression occurs by two different mechanisms. First, cooperative interactions between two DNA-bound proteins can be modulated by the binding of a third protein. Second, the binding of a protein to its cognate sequence can be modulated by the binding of another protein. These results demonstrate that the interactions between the proteins in a multicomponent nucleoprotein complex cannot be described by simple pairwise cooperativity models. This thesis suggests that transcriptional activation and repression cannot be studied as independent mechanisms, and provides a first step toward understanding the interplay of DNA structure, protein-protein and protein-DNA interactions, which together regulate the formation and efficiency of a transcription initiation complex.

Description

Keywords

Biochemistry., Biophysics., Genetics.

Citation

Source: Dissertation Abstracts International, Volume: 56-04, Section: B, page: 1990.;Advisors: Michael Brenowitz.