Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

Intrinsically curved DNA upstream of promoters was shown to affect the Bacillus subtilis RNA polymerase-promoter interactions leading to transcription initiation. The Alu156, a B. subtilis bacteriophage dependent on the curved DNA located immediately upstream of the $-$35 region, and modified forms of Alu156 were used. Promoters were examined using gel retardation and DNase I footprinting assays. DNase I footprinting analysis of the Alu156 promoter demonstrated a unique pattern of DNase I hypersensitive and protected sites characteristic of DNA wrapped around the protein. This wrapping was observed upstream of the curved DNA. To examine the nature of curved DNA, the native curved DNA in Alu156 was replaced with synthetic curved DNA containing one, two, or three adenine tracts (Alu1A, Alu2A, or Alu3A). While the levels of binding to RNA polymerase, DNA wrapping and transcription were greater in the Alu3A promoter than Alu1A and Alu2A promoters, the levels never equaled those observed with Alu156. Since the native curved DNA of Alu156 is highly rich in (A+T) and (A+T)-rich DNA immediately upstream of the promoter is known to function as an $\alpha$ binding site, promoters containing (A+T)-rich regions were examined. These promoters displayed a significant increase in promoter activity relative to promoters lacking the curved DNA but never equaled the original Alu156 promoter. These results suggested that both the (A+T)-content and curvature of the native curved DNA of Alu156 contribute to RNA polymerase binding and transcription. Using a combination of temperature and competitor DNA to control which transcription intermediates were formed, the RNA polymerase-promoter complex that was wrapped was identified as an unstable, closed promoter complex analogous to the RP$\rm\sb{c1}$ complex. These studies also identified a relatively stable, closed complex that lacked DNA wrapping and met the criteria for RP$\rm\sb{c2}.$ Based on DNase I footprinting and transcription assays, the presence of the curved DNA on Alu156 greatly stimulated the formation of the RP$\rm\sb{c1}$ and its subsequent isomerization to RP$\rm\sb{c2}.$.

ISBN

9780599081802

Pages

122

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