Escherichia coli RNA polymerase core and holoenzyme structures

Multisubunit RNA polymerase is an essential enzyme for regulated gene expression. Here we report two Escherichia coli RNA polymerase structures: an 11.0 A structure of the core RNA polymerase and a 9.5 A structure of the sigma(70) holoenzyme. Both structures were obtained by cryo-electron microscopy and angular reconstitution. Core RNA polymerase exists in an open conformation. Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in ss'. All common RNA polymerase subunits (alpha(2), ss, ss') could be localized in both structures, thus suggesting the position of sigma(70) in the holoenzyme.     

RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex

Using high-resolution atomic force microscopy (AFM) we show that in a ternary complex of an activator protein, FIS, and RNA polymerase containing the sigma(70) specificity factor at the Escherichia coli tyrT promoter the polymerase and the activator form discrete, but connected, subcomplexes in close proximity. This is the first time that a ternary complex between an activator, a sigma(70) polymerase holoenzyme and promoter DNA has been visualised. Individually FIS and RNA polymerase wrap approximately 80 and 150 bp of promoter DNA, respectively. We suggest that the architecture of the ternary complex provides a general paradigm for the facilitation of direct, but weak, interactions between polymerase and an activator.     

Identification of close contacts between the sigma N (sigma 54) protein and promoter DNA in closed promoter complexes.

The complexes forming between the alternative sigma factor protein sigma N (sigma 54), its holoenzyme and promoter DNA were analysed using the hydroxyl radical probe and by photochemical footprinting of bromouridine-substituted DNA. Close contacts between the promoter, sigma N and its holoenzyme appear to be restricted predominantly to one face of the DNA helix, extending from -31 to -5. They all appear attributable to sigma N and no extra close contacts from the core RNA polymerase subunits in the holoenzyme-promoter DNA complex were detected. We suggest that the apparent absence of close core RNA polymerase contacts in the region of the promoter DNA to be melted during open complex formation is important for maintaining the closed complex. Results of the hydroxyl radical footprinting imply that sigma N makes multiple DNA backbone contacts across and beyond the -12, -24 consensus promoter elements, and the photochemical footprints indicate that consensus thymidine residues contribute important major groove contacts to sigma N. Formation of the open complex is shown to involve a major structural transition in the DNA contacted by sigma N, establishing a direct role for sigma N in formation of the activated promoter complex.     

Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex

We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding sigma(70)-(RNA polymerase core), sigma(70)-DNA, and sigma(70)-RNA interactions. The positions of sigma(70) regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of sigma(70) region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a "molecular mimic" of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.     

Mapping protease susceptibility sites on the Escherichia coli transcription factor sigma70.

N-terminally and C-terminally histidine-tagged versions of Escherichia coli RNA polymerase initiation factor sigma70 were subjected to limited proteolysis and electrophoretic separation. The protein fragments were transferred to nitrocellulose, and biotinylated nitrilotriacetic acid was used to detect the His-tagged ladder that resulted. Using size markers of known lengths derived from chemical cleavage of the same His-tagged sigma70, we were able to map the sites of proteolysis for sigma70 free in solution, bound to core RNA polymerase, and in the Mg2+-dependent open complex with lambdaPR promoter DNA. Numerous sites of changed susceptibility were mapped. Most of these sites mapped near residues 100 and 500. In addition, the highly acidic region around residue 190 became susceptible to cleavage in the open promoter complex. These results suggest that sigma70 undergoes significant conformational changes upon binding to core RNA polymerase and upon open promoter complex formation.