Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

Regulation of transcription most often occurs at the stage of initiation. RNA polymerase binding to the transcription start site, the promoter, is influenced by many nucleotide sequence elements. The predominant recognition sequences are those bound by the $\sigma$ subunit of RNA polymerase located at $-$10 and $-$35 relative to initiation site of most promoters. Another element involved in this regulation is intrinsic DNA curvature. In this study, the contribution of intrinsically curved DNA immediately upstream of the promoter to the interaction between Escherichia coli RNA polymerase and this DNA was examined. DNase I footprinting analysis confirmed that RNA polymerase wraps DNA upstream of the promoter around the enzyme. The nature of interaction between DNA upstream of promoter and RNA polymerase was explored using addition of NaCl. The wrapped complex was not observed at NaCl concentration above 150 mM suggesting the electrostatic sequence-independent nature of the interaction. Study of the effect of temperature on DNA wrapping and open promoter complex formation demonstrated the existence of closed, wrapped complexes. No wrapped complexes survived a 30 second heparin challenge indicating the absence of wrapped open complexes. The above data provide evidence that DNA wrapping occurs prior to open complex formation. Promoters containing an AT-rich region or the UP element of ribosomal RNA promoter rnBPI were constructed. Using a gel retardation assay, the relative affinity of RNA polymerase for these promoters was compared to that for curved DNA-containing promoter. The promoter containing curve DNA displayed the highest binding to RNA polymerase. The presence of curved DNA favored the formation of the wrapped complex. A run-off transcription assay limited to a single round of initiation examined the efficiency of transcription for these promoters as a function of temperature. Relative to promoters lacking curved DNA, the promoter with curved DNA formed significantly more heparin-resistant, closed complexes at low temperature. These complexes could quickly isomerize to open complex at 37$\sp\circ$C. We proposed that curved DNA facilitates wrapping of DNA around RNA polymerase and enhances the transition from a heparin sensitive closed complex to a heparin resistance closed promoter complex.

ISBN

9780591723915

Pages

99

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