Activation of transcription by protein-induced DNA bending: Evidence for a DNA structural transmission model

This dissertation characterizes the role of Integration Host Factor (IHF) and negative DNA supercoiling in the regulation of transcription initiation of the ilvGMEDA operon of Escherichia coli. IHF is a DNA bending protein that binds to an upstream activating sequence (UAS1) 92 base pairs from the transcriptional initiation site and, on a negatively supercoiled DNA template, enhances transcription from the ilvP{dollar}rmsb{lcub}G{rcub}{dollar} promoter of the ilvGMEDA operon of Escherichia coli. Transcriptional activation is maintained when the orientation of the upstream IHF binding site is reversed, the opposite face of the nonsymmetrical, heterodimeric, IHF molecule is presented to the downstream RNA polymerase. Additionally, IHF can be functionally replaced by a heterologous mammalian DNA bending protein, Lymphoid Enhancer Binding Factor (LEF-1). These observations argue against activation by specific protein-protein interactions and suggest a role for DNA bending in the activation mechanism.; Chemical probes of DNA structure were employed to show that IHF binding at UAS1 is accompanied by an unwinding of the DNA helix in the {dollar}-{dollar}10 region of the ilvP{dollar}rmsb{lcub}G{rcub}{dollar} promoter. In vitro, abortive transcription, assays performed on closed circular, negatively supercoiled DNA templates showed that IHF increases the rate of RNA polymerase isomerization from a closed to an open complex. These results suggest that the IHF-mediated DNA bend induces a partial unwinding of the DNA helix that increases the rate of the isomerization step of the transcription initiation reaction.; The in vivo and in vitro influence of DNA supercoiling on transcription from the ilvP{dollar}rmsb{lcub}G{rcub}{dollar} promoter was also examined. In the absence of IHF, ilvP{dollar}rmsb{lcub}G{rcub}{dollar} promoter activity increases with increased DNA supercoiling. In the presence of IHF, on supercoiled DNA template, a 5-fold greater response to increasing DNA supercoiling is observed. However, the relative transcriptional activities of the promoter in the presence and absence of IHF at any given DNA superhelical density remains the same. Therefore, IHF and increased DNA supercoiling activate transcription by different mechanisms. IHF binding does not activate transcription by differential binding to relaxed and supercoiled DNA templates since IHF binds with equal affinities to its target site on linear and supercoiled DNA templates.