Sigma38 (rpoS) RNA polymerase promoter engagement via -10 region nucleotides

Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase. Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage. However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong. This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (-12T and -11A) being of paramount importance. A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the -10 region (CTAcacT from -13 to -7). The remaining two nucleotides were shown to have different roles, -13C in preventing recognition by the heterologous sigma70 polymerase and -7T in directing enzyme isomerization. These -10 region nucleotides appear to have their primary function prior to full melting because probes that had a melted start site were relatively insensitive to substitution at these positions. These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.     

Conformation of fork junction DNA in a complex with Escherichia coli RNA polymerase

Escherichia coli RNA polymerase is able to bind fork junction DNA containing a conserved -10 promoter element in a sequence-specific manner, and it is believed that polymerase-fork junction DNA interaction mimics those between the enzyme and the promoter DNA in the open complex. In this report we determined the conformation of polymerase-bound fork junction DNA in solution. A series of distances between sites in the fork junction DNA in complex with polymerase were determined using luminescence and fluorescence resonance energy transfer. A series of fork junction DNAs were prepared containing the luminescent or fluorescent donor probe at the upstream or at the downstream end of the fork DNA and acceptor probes at nine positions within the fork junction DNA. The measured distances were compared with analogous distances in a model reference DNA duplex, and the observed distance differences were used to build a model of the fork junction DNA in a complex with the polymerase. The obtained model revealed an insignificant perturbation of the duplex part of the fork DNA in a complex with the polymerase whereas a sharp kink of DNA was observed at the ds/ss DNA boundary of the fork junction DNA.     

Open complex formation in vitro by sigma38 (rpoS) RNA polymerase: roles for region 2 amino acids

Non-functional mutants of sigma(38)(sigma(S)) were studied in vitro to identify the nature of their defects. Mutations in four amino acids led to severe defects in DNA binding and enzyme isomerization with promoter fork junction probes containing single-stranded non-template DNA. The same properties were previously seen with DNA mutations at the fork junction, implying that sigma:DNA interactions at the fork junction are used both for DNA binding and enzyme isomerization. An overlapping set of four mutants had defects that appear to be associated with DNA melting to create the fork junction. When mapped onto the sigma(70) structure, these groups of mutants suggest motifs used by sigma factors to melt DNA and isomerize RNA polymerase to form functional open promoter complexes.     

Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA.

Sequence analysis of 236 promoters recognized by the Bacillus subtilis sigma A-RNA polymerase reveals an extended promoter structure. The most highly conserved bases include the -35 and -10 hexanucleotide core elements and a TG dinucleotide at position -15, -14. In addition, several weakly conserved A and T residues are present upstream of the -35 region. Analysis of dinucleotide composition reveals A2- and T2-rich sequences in the upstream promoter region (-36 to -70) which are phased with the DNA helix: An tracts are common near -43, -54 and -65; Tn tracts predominate at the intervening positions. When compared with larger regions of the genome, upstream promoter regions have an excess of An and Tn sequences for n > 4. These data indicate that an RNA polymerase binding site affects DNA sequence as far upstream as -70. This sequence conservation is discussed in light of recent evidence that the alpha subunits of the polymerase core bind DNA and that the promoter may wrap around RNA polymerase.