The mechanism of transcription activation by the bacillus subtilis response regulator, SpoOA

In Bacillus subtilis, signals which indicate nutrient depletion initiate a complex series of events leading to endospore formation. Environmental signals are detected by multiple sensor kinases and a cellular response is elicited by phosphorylation of the transcription regulator, Spo0A, which initiates endospore formation by specifically stimulating or repressing expression of developmental genes. In this thesis, the in vitro properties of Spo0A have been examined using the promoter for the spollG operon as a target. In vivo expression from the spollG promoter is absolutely dependent on phosphorylated Spo0A and RNA polymerase containing the major sigma subunit, σA . As a first step, a comparison of the activation characteristics of phosphorylated Spo0A (Spo0A~P) and a constitutively active form of Spo0A consisting of the C-terminal 124 amino acids (Spo0ABD) was carried out. Both Spo0A~P and Spo0ABD stimulated transcription from the spollG promoter 10 fold more efficiently than Spo0A. DNAse I footprint assays revealed that phosphorylation enhanced binding of intact Spo0A to the 0A boxes, while the binding of Spo0ABD was similar to that of Spo0A. Thus, the activation of Spo0A is not primarily due to enhanced DNA binding. Experiments testing the effects of ionic strength indicated that the presence of a phosphorylated N-terminus increased the stability of Spo0A-RNA polymerase complexes at the spollG promoter. Potassium permanganate probing revealed that when activated Spo0A was incubated with RNA polymerase and a DNA fragment containing the spollG promoter, base pairs between -13 and -3, relative to the start site of transcription, were denatured. Bases downstream of -3 were exposed in the presence of initiating nucleotides. Addition of activated Spo0A or RNA polymerase alone did not induce denaturation. Heteroduplex templates that contained the non-template sequence of the wild type promoter on both strands between positions -3 and -13 were efficiently transcribed without activated Spo0A. These data suggested that DNA strand separation was a two step process and that the activation of Spo0A created a form that interacted with the polymerase to induce the first of the two steps. In vitro transcription from the spollG promoter by Bacillus subtilis RNA polymerase reconstituted with wild type alpha subunits or with C-terminal deletion mutants of alpha was examined. Wild type or mutant polymerases were stimulated by activated Spo0A although some differences in the interaction of the enzymes with the DNA were noted. Mutational analysis of the C-terminus of Spo0A identified 3 residues required for activation of the spollG promoter. These and other data support a proposal in which activation of Spo0A triggers a structural change in the regulator that exposes a RNA polymerase interaction domain. Contacts between activated Spo0A and RNA polymerase induce the upstream denaturation event and it is proposed that stimulation of the spollG promoter by Spo0A occurs by accelerating this denaturation step. [Scientific formulae used in this abstract could not be reproduced.]Science, Faculty ofMicrobiology and Immunology, Department ofGraduat