| Bacterial RNA polymerase is a highly regulated enzyme, altering the metabolic and functional state of bacteria in response to their environment. rRNA synthesis is a major metabolic expense and is, thus, highly regulated. One such response is the ppGpp-induced stringent response which changes the metabolic state of the cell, particularly inhibiting rRNA synthesis. ppGpp, guanosine tetraphosphate, is produced rapidly during amino acid starvation. ppGpp acts along with an accessory molecule, a protein DksA. The mechanism and binding site of ppGpp/DksA remains unclear and disputed. The objective of this study is to determine the binding site of ppGpp and DksA and understand the mechanism by which they regulate the cell's metabolic state. The strategy is to randomly mutagenize RNAP genes, specifically rpoC which codes for beta subunit, and perform genetic screens to identify mutants resistant to toxic effects of ppGpp/DksA. The mutants were isolated, sequenced, tested and characterized by in vivo and in vitro assays. The mutants isolated in this study belong to two distinct clusters: the switch region, which controls the opening and closing of active center clamp allowing dsDNA entry, and the secondary channel, which is the channel through which NTP substrates enter. The mutants in the switch region could imply a conformational determinant for ppGpp/DksA. The secondary channel mutants could be binding/conformational determinants for ppGpp/DksA. The in vitro results indicate that two mutants in the secondary channel interact with DksA which could confirm DksA's putative binding site. |
