A detailed analysis of the DNA binding properties and the affinity purification of the Drosophila heat-shock transcription factor

Biochemistry and Molecular Biophysics

Shuey, David John ; Parker, Carl Stevens

University:California Institute of Technology

Department:Chemistry and Chemical Engineering

关键词: Biochemistry and Molecular Biophysics;   

Others  :  https://thesis.library.caltech.edu/6800/1/Shuey_dj_2002.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

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 researchefforts 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 transcriptionalactivator 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.