| Escherichia coli DksA is a non-classical transcription factor that binds directly to RNA Polymerase (RNAP) and not to DNA, giving DksA the potential to influence transcription from all promoters. However, because DksA functions by destabilizing transcription initiation complexes, it only inhibits transcriptional output from those promoters that make unstable complexes with RNAP. In E. coli, most RNAP-promoter complexes are long-lived. The effects of DksA on promoters that form short-lived complexes are amplified by the "alarmone" ppGpp, which also binds directly to E. coli RNAP. DksA and ppGpp are most known for inhibition of rRNA synthesis, but these factors can also activate or repress transcription from an additional ∼700 genes in E. coli, directly or indirectly.;The goal of my work was to better understand the molecular mechanism employed by DksA. First, to more precisely dissect the role of DksA and RNAP substitutions on DksA function, I describe an assay for quantitative measurement of the affinity of DksA for RNAP. This assay has allowed us to determine whether substitutions in DksA or RNAP that reduce DksA effects are a result of decreased binding affinity or a loss of function at a step after binding. Next, by site-specific incorporation of the cross-linkable amino acid benzophenylalanine into DksA, I established physical constraints between DksA and RNAP that made it possible to employ a molecular docking program (HADDOCK) to generate a model of the DksA-Core RNAP complex. I subsequently established that a direct contact can form between the coiled-coil tip of DksA and the mobile trigger loop domain of RNAP, that this contact is required for RNAP to respond to DksA, and along with previous work, I propose a detailed mechanism of DksA mediated negative regulation. Finally, I examined the role of a DksA-like protein (RSP2654) and ppGpp from Rhodobacter sphaeroides and show that RSP2654 and ppGpp directly destabilize transcription initiation complexes, suggesting a conserved mechanism of action in gamma-(E. coli) and alpha-proteobacteria (R. sphaeroides). |
