Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

The major RNA polymerases of Bacillus subtilis and Escherichia coli recognize the same conserved sequences at $-$10 and $-$35 of the promoter. The B. subtilis enzyme requires additional DNA sequences for efficient promoter utilization. Deletion mutants were used to demonstrate that the function of two B. subtilis phage SP82 promoters, Ba1129 and Alu156, was dramatically affected by sequence upstream from $-$35. The upstream DNA contained runs of four to six adenines with a 10-11 base pair periodicity. Indicative of DNA curvature, these regions decreased electrophoretic mobility of DNA fragments containing either promoter. The curved upstream DNA increased the affinity of both promoters for RNA polymerase. This resulted in increased in vivo transcription from Alu156 but decreased transcription from Ba1129. The extremely stable RNA polymerase:DNA complexes formed with the Ba1129 promoter suggested that transcription from this promoter may be limited at promoter clearance following initiation. Transcription from the Alu156 promoter was limited at the level of RNA polymerase binding and strongly dependent on the curved upstream DNA. Alu156 mutant promoters were constructed by insertion of oligonucleotides between the $-$35 region and the curved DNA to examine the structure/function relationship of these two regions. The incremental insertions altered the rotational orientations as well as the distance between the promoter and upstream curved DNA. In vivo expression, in vitro transcription, and RNA polymerase binding were all modulated by the rotational orientation. The most efficient mutant promoters were those in which the original orientation was preserved. The electrophoretic mobilities of these mutants suggested that the upstream curve directionally opposes a downstream curve, conferring an S shape to the DNA of this promoter. These results are consistent with a model in which the RNA polymerase either directly contacts the curved DNA resulting in additional protein-DNA interactions through a wraparound effect or contacts DNA upstream from the polyadenine tracts using a curvature-mediated DNA looping mechanism. The Ba1129 and Alu156 upstream regions were used to increase in vivo expression from the lambda phage P$\sb{\rm L}$ and P$\sb{\rm R}$ promoters in B. subtilis. No correlation was observed between RNA polymerase binding and expression for the hybrid lambda promoters.

Pages

158

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