Investigating the Kinetics of HSF Binding Using High Throughput Single Molecule Imaging

Abstract

Transcriptional regulation in the heat shock response is vital for the proper function and survival of both prokaryotic and eukaryotic cells. The bulk of our current understanding of transcription has been obtained through biochemical assays such as pull-down, knockdown and genome wide assays. These have provided information on the identity of proteins involved in the regulation of various genes and the location at which they bind to DNA. However, the kinetics and architecture are not known at a single molecule level, in part due to the difficulty of obtaining enough data to provide statistics. A flow cell designed to extend DNA molecules under a hydrodynamic force, nicknamed DNA curtains, provides a high throughput method of imaging protein-DNA interactions at the single molecule level. Electron beam lithography is the traditional method used to pattern these flow cells, which is a cost prohibitive process. We have shown that a simpler and less costly method, photolithography, can instead be used, therefore enabling this technique to be more widely available. Using these DNA curtains, we will investigate the details of heat shock factor (HSF) DNA binding, cooperativity, and target searching at a single molecule level.