Doctor of Philosophy (PhD)
Adenine-tract mediated, intrinsic DNA curvature is found upstream of highly transcribed promoters in bacteria. In Bacillus subtilis, curved DNA immediately upstream of the core promoter stimulates promoter function. The curved DNA functions in a position-sensitive fashion indicating that it likely interacts with the RNA polymerase. There are two components of the curved DNA that mediate this interaction with RNA polymerase, the A+T rich nature of the runs of adenines spaced with the helical repeat of the DNA and the curved shape of the DNA. The alpha subunit of the RNA polymerase from Escherichia coli is known to bind to A+T rich DNA. As predicted from the conservation of the amino acid sequences for E. coli and B. subtilis alpha subunits, the B. subtilis alpha subunit bound the adenine tracts of the curved DNA. The shape of curved DNA promotes DNA structuring in which the DNA is wrapped about a protein. Since B. subtilis RNA polymerase is known to wrap the DNA about the enzyme, curved DNA would favor this wrapping. To determine the role of alpha subunit binding in the enhancement of transcription initiation by curved DNA, the DNA binding domain of B. subtilis alpha subunit was deleted. With a deletion of 36 amino acids from the carboxyl terminal end of alpha, the ability to bind A+T rich DNA sequences was abolished and the ability of RNA polymerase containing this alpha subunit to be affected by the curved DNA was significantly reduced. While promoters with curved DNA were transcribed more effectively than those with A+T rich DNA, a functional alpha was necessary for the curved DNA to fully stimulate transcription initiation. With a deletion of 59 amino acids from alpha, the subunit lost sequence recognition but demonstrated strong non specific DNA binding. Reconstituted RNA polymerase containing this truncated alpha subunit failed to exhibit significant transcription activity but generated a normal pattern of promoter binding by DNase I footprinting. This indicated that the interaction of the alpha subunit with other subunits in the enzyme during transcription initiation was impaired by the deletion of 59 amino acids from the carboxyl terminus of alpha.
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Telles, Caroline, "The contribution of the alpha subunit of the Bacillus subtilis RNA polymerase in the enhancement of transcription by curved DNA" (2003). LSU Doctoral Dissertations. 2004.