UBC Theses and Dissertations
SPO0A repression of transcription from the bacillus subtilis AbrB promoter is mediated by SPO0A tethering RNA polymerase to the promoter region Greene, Elizabeth Anne
Bacillus subtilis forms an endospore as a response to nutritional stress. Spore formation produces only one progeny cell, therefore it is advantageous for cells to avoid sporulating if other mechanisms for survival are available. The abrB gene product is involved in repressing sporulation. Transcription of abrB is repressed by the SpoOA protein. SpoOA is present in the cell at low levels during vegetative growth, and is activated by phosphorylation. The purpose of this thesis was to define the mechanism by which phosphorylated SpoOA represses abrB transcription. There are two SpoOA concensus binding sites (OA boxes) downstream from the abrB promoter and a third OA box upstream from the promoter. Based on this structural information, three models for SpoOA(-P) mediated inhibition of abrB transcription were developed. In the first model a DNA loop is formed by protein:protein interactions between SpoOA(-P) bound at the upstream and downstream OA boxes, blocking RNA polymerase binding at the abrB promoter. The second model proposed that SpoOA(-P) binds the downstream OA boxes, blocking RNA polymerase from binding to the abrB promoter region. In the third model RNA polymerase and SpoOA(-P) bind the abrB promoter region simultaneously. SpoOA(-P) prevents transcription from proceeding by "tethering" polymerase at the promoter. The DNA looping model was tested by measuring SpoOA(-P) repression of abrB transcription on promoter fragments with or without the upstream OA box. Transcription of abrB was inhibited in the absence of the upstream OA box, therefore DNA looping between the upstream and downstream OA boxes was not necessary for repression of abrB transcription. To determine whether RNA polymerase and Spo0A(~P) bound to the abrB promoter simultaneously, the ability of the two proteins to protect abrB DNA from cleavage by DNasel or hydroxyl radical was explored. If binding of RNA polymerase and SpoOA(-P) were found to be mutually exclusive then the blocking model would be the most accurate, however, both proteins protected abrB promoter regions simultaneously therefore RNA polymerase was shown to be tethered at the abrB promoter by SpoOA(-P).