Construction and characterization of Streptomyces coelicolor A3(2) mutants that are multiply deficient in the nonessential hrd-encoded RNA polymerase sigma factors.

Previous studies showed that Streptomyces coelicolor A3(2) has four genes (hrdA, hrdB, hrdC, and hrdD) that appear to encode RNA polymerase sigma factors very similar to the sigma 70 subunit of Escherichia coli and that hrdC and hrdD could be individually disrupted without causing obvious phenotypic defects. Here, hrdA was cloned and stable null hrdA and hrdD mutants were constructed by gene replacement. These two mutants and a previously constructed hrdC null mutant were used in crosses to generate hrdAC, hrdAD, hrdCD, and hrdACD strains. The inability to synthesize one, two, or all three of the nonessential hrd-encoded sigma factors had no obvious phenotypic consequences.     

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.     

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.     

Purification and properties of RNA polymerases from mother cells and forespores of sporulating cells of Bacillus subtilis

Bacillus subtilis sporulating cells at stage III were fractionated into mother cell and forespore fractions by means of a lysozyme-detergent method. Three forms of DNA-dependent RNA polymerase enzymes, termed M sigma, F sigma, and F delta, in addition to core enzyme (alpha 2, beta', and beta) have been purified from the cell fractions. Enzymes M sigma and F sigma are present in the mother cell and forespore, respectively, and contain sigma factor of 55,000 daltons in addition to the core subunits. On the other hand, enzyme F delta is present specifically in the forespore and contains delta 1 factor of 28,000 daltons instead of the sigma factor. The amount of RNA polymerase in the forespore is about twice that in the mother cell. The enzymes M sigma and F sigma also differed in their elution profiled from DEAE-cellulose columns and in their heat stabilities indicating that the two sigma-containing holoenzyme forms may be different in their structural properties. The enzyme F delta transcribed B. subtilis DNA about 1.6 times more actively than enzyme F sigma, and the enzymes M sigma and F sigma transcribed the DNA about 2.2 times more actively than did core enzyme.     

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.     

Interaction of Escherichia coli RNA polymerase with eukaryotic TATA-binding protein

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.