| Bacterial RNA polymerase (RNAP) is a multi-subunit (&agr; 2, beta, beta', &ohgr;, and sigma) enzyme that synthesizes RNA from a DNA template. My research has focused on the mechanism of formation of transcriptionally-competent open promoter complexes by E. coli RNAP, using the lambdaPR promoter as a model system. Using chemical footprinting and filter binding techniques, I have tested our mechanistic proposals that RNAP bends duplex promoter DNA into the active site cleft, opens 13 DNA base pairs in the cleft and loads the +1 base of the template strand into the active site, and then assembles and tightens several mobile protein elements on downstream duplex DNA as the discriminator region (-6 to -2) of the nontemplate strand rearranges.;My fast permanganate and hydroxyl radical footprinting data demonstrate that the first kinetically significant intermediate I1 is unambiguously a closed complex in which the downstream duplex has been inserted in and is periodically protected by the RNAP cleft; further, the nontemplate strand of the I2 intermediate is globally less protected than in the final open complexes. Two RNAP variants (one a partial deletion of the beta' jaw (DeltaJAW), and the other a deletion of region 1.1 of sigma70 (Delta1.1)) are found to associate with lambdaPR promoter DNA slower than wild-type (WT) RNAP complexes. My hydroxyl radical footprints and dissociation kinetic studies on DeltaJAW at full length or downstream-truncated promoter DNA indicate that the jaw forms an interface with the far-downstream duplex (+10 to +20) after DNA opening. Urea-dependence dissociation kinetic studies show that folding/assembly of the jaw contributes to stabilization of the late steps of open complex formation, likely in the conversion of I 3 to RPo. Kinetic dissociation studies indicate that the lifetime of the Delta1.1 open complex is greatly extended relative to WT RNAP. Heparin increases the dissociation rate of Delta1.1 but not WT open complexes. Furthermore, at high heparin concentrations the dissociation rate of Delta1.1 exceeds that of WT open complexes, suggesting that region 1.1 of sigma70 is required to displace one or both strands of the initiation bubble in the cleft in dissociation of WT open complexes. |
