Regulating a Paused Polymerase: The Role of Heat Shock Factor, and the Correlation Between Polymerase Pausing and Synchronous Patterns of Transcriptional Induction
Open Access
Author:
Stefanik, Laura Michelle
Graduate Program:
Genetics
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
March 27, 2012
Committee Members:
David Scott Gilmour, Thesis Advisor/Co-Advisor
Keywords:
RNA Pol II Pausing Heat Shock Factor HSF Transcription Synchrony Promoter
Abstract:
The regulation of gene expression can occur at multiple points along the pathway of transcription and translation. One mechanism of regulating gene expression levels involves the pausing of RNA Polymerase II (RNA Pol II) on genes by the protein complexes DSIF and NELF. RNA Pol II is then poised to rapidly transcribe the genes in response to an appropriate stimulus. On the heat shock protein (hsp) genes in Drosophila melanogaster, the transcriptional activator Heat Shock Factor (HSF) senses cellular stress such as heat shock and reactivates a paused RNA Pol II to a state of active transcription in a process that is collectively known as the heat shock response. In addition to the heat shock response, the biological importance of Pol II pausing is evident from its correlation with genes that are synchronously induced in an organized fashion during development.
This thesis discusses the purification of recombinant Drosophila melanogaster HSF to make a polyclonal antibody against it for the purpose of using the antibody in various biochemical assays, including electrophoretic mobility shifts and in vitro transcription reactions. The second part of the thesis analyzes the contribution of the minimal promoter on transcriptional activation patterns and the presence of a paused polymerase.
