A Detailed Analysis of the DNA Binding Properties and the Affinity Purification of the Drosophila Heat-Shock Transcription Factor

Citation

Shuey, David John (2002) A Detailed Analysis of the DNA Binding Properties and the Affinity Purification of the Drosophila Heat-Shock Transcription Factor. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/y91j-s092. https://resolver.caltech.edu/CaltechTHESIS:01312012-161653671

Abstract

The heat shock response presents an extremely attractive model to study the regulation of gene transcription in eucaryotes. I have focused the bulk of my research efforts on investigating the molecular determinants of protein-DNA interaction exhibited by the heat-shock transcription factor (HSTF) of Drosophila. The specific "contacts" made by the HSTF upon binding to the heat-shock element (HSE) were exhaustively determined using a variety of chemical and enzymatic probes for a number of HSTF-HSE complexes formed at both hsp70 and hsp83 gene promoters. During the course of these studies it is demonstrated that the HSTF appears to polymerize in a sequence-specific and template-directed manner on each of these promoters, a novelty in this class of regulators. Evidence suggesting that DNA bending may occur during this HSTF-DNA association on the hsp70 promoter is also presented. This observation represented the first report of a eucaryotic transcriptional activator exhibiting this property.

Significantly, during the course of these studies two novel technologies were advanced; namely the gel-based contact point method and sequence-specific DNA affinity purification methodology. These technical advancements and the HSTF-HSE interaction results will be discussed in the body of the thesis in light of their relevance to both heat-shock and global gene expression mechanisms.

Thesis Files