Date of Award

1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

Many promoters contain a curved DNA component essential for high levels of transcription activity. The present work approached the study of these unusual DNA structures in a twofold manner. First, promoter binding by RNA polymerase from Bacillus subtilis and Escherichia coli was examined using a collection of promoters containing DNA curvature. Promoter binding by both RNA polymerases was governed primarily by the nucleotide sequence at the highly conserved $-$10 and $-$35 regions of the promoters. However, the presence of curved DNA, immediately upstream of the $-$35 region of the promoter, predictably increased binding to those promoters which contained curved DNA by the B. subtilis RNA polymerase. Binding by the E. coli RNA polymerase was modestly affected by DNA curvature. The second approach was to characterize the mechanism involved in transcription stimulation by this curve or intrinsically bent DNA. Generally, the proposed mechanisms for transcription initiation include enzyme conformational changes leading to strand separation. Formation of RNA polymerase-promoter complexes that entail significant conformational changes is sensitive to changes in temperature. A goal in the second part of this work was to test if curved DNA influenced a step in transcription initiation that was sensitive to temperature changes. The formation of open promoter complexes was measured using a B. subtilis phage promoter containing curved DNA upstream of the $-$35 region, the Alu156 promoter, and mutants of Alu156 in which the curved DNA sequences were displaced upstream. Open promoter complexes were measured by (1) a run-off transcription assay limited to a single round of initiation and (2) the direct detection of single stranded DNA using potassium permanganate cleavage. Promoters with properly aligned curved DNA formed open promoter complexes at lower temperatures than promoters with misaligned curved DNA. Also, curved DNA enhanced formation of open promoter complexes as measured by actual strand separation. A model describing the effect of curved DNA on open promoter complexes was proposed.

Pages

128

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