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Spo0A-stimulated transcription initiation at the bacillus subtilis spoIIG promother Seredick, Stephen David

Abstract

As a last resort to insurmountable environmental stresses, the common soil bacterium Bacillus subtilis undergoes a complicated morphological transformation to produce a metabolically dormant spore. The central regulator of sporulation is a response regulator transcription factor, Spo0A. When activated by phosphorylation, Spo0A~P directly stimulates or represses transcription at a number of promoters encoding genes involved in the early stages of sporulation. This thesis explores the mechanism by which Spo0A~P activates expression at one of these promoters, the spoIIG promoter. At the spoIIG promoter, Spo0A~P binds to 0A boxes and interacts with RNA containing σ[sup A] to facilitate DNA strand separation. Precisely how Spo0A~P stimulated DNA strand separation was not understood. In this thesis, I show that Spo0A~P acts prior to denaturation of the DNA strands and has no effect on transcription from DNA templates in which the -10 element is artificially denatured to expose the non-template strand. Investigation of the thermal dependence of transcription from a series of artificially denatured promoters suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A~P did not reduce this energetic barrier. Kinetic assays revealed that Spo0A~P stimulated both the rate of formation of initiated complexes as well as increasing the number of RN AV-spoIIG complexes capable of initiating transcription. These results implied that Spo0A exerted its effect on RNA polymerase prior to the formation of an open complex. I present evidence for a closed intermediate complex formed by RNA polymerase, Spo0A~P and the spoIIG promoter. To isolate the effect of Spo0A~P on events prior to DNA strand separation at the spoIIG promoter I used DNA fragments that contained only promoter sequences 5' to the -10 element in electrophoretic mobility shift assays. RNA polymerase bound to these fragments readily and the RNA polymerase- spoIIG complexes recruited Spo0A~P. This complex was stabilized by Spo0A~P. In addition I investigated the effect of truncating the upstream portions of the spoIIG promoter which contain a pair of Spo0A binding sites at approximately -90 relative to the transcription start site and found that this DNA inhibited the binding of RNA polymerase. Strikingly, Spo0A~P binding depended on the presence of the DNA upstream of the 0A boxes, suggesting a role for the RNA polymerase a subunit carboxy-terminal domains in transcription at this promoter. Finally, Spo0A~P binding to the promoter played two distinct roles. Promoter-distal bound Spo0A~P increased the local concentration of Spo0A~P available to bind to promoter-proximal Spo0A binding sites, while promoter-proximal bound Spo0A~P stabilized and modified pre-formed RNA polymerase-spoIIG complexes. The data is consistent with a model whereby Spo0A~P stabilizes RNA polymerase during the initial stages of open complex formation at the spoIIG promoter, but only until the nontemplate strand of the -10 element is fully single-stranded. These effects account for transcription activation of the spoIIG promoter by Spo0A~P.

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