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.     

Specific recognition of the -10 promoter element by the free RNA polymerase sigma subunit

Bacterial RNA polymerase holoenzyme relies on its sigma subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the sigma subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free sigma, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with sigma region 1 playing a role in the masking. To analyze the DNA-binding properties of the free sigma, we selected single-stranded DNA aptamers that are specific to primary sigma subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended -10 promoter. We demonstrate that recognition of this motif by sigma region 2 occurs without major structural rearrangements of sigma observed upon the holoenzyme formation and is not inhibited by sigma regions 1 and 4. Thus, the complex process of the -10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated sigma subunit and a short aptamer oligonucleotide.     

Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.     

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.     

Differences in Contacts of RNA Polymerases from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Thermus aquaticus</Emphasis> with <Emphasis Type="Italic">lac</Emphasis>UV5 Promoter Are Determined by Core-Enzyme of RNA Polymerase

The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.