Coupling σ Factor Conformation to RNA Polymerase Reorganisation for DNA Melting

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (σ⁵⁴-RNAP, Eσ⁵⁴) and a slowly hydrolysed ATP analogue (ATPγS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of σ⁵⁴-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Eσ⁵⁴ occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity σ⁵⁴ factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic β/β′ subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Eσ⁵⁴ promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the β′ jaw domain, thereby allowing acquisition of the open complex status.     

Inhibitory effect of phosphate on in vitro development of 2-cell rat embryos is overcome by a factor(s) in oviductal extracts

The promoter recognition site on the sigma70 initiation factor is shielded from interaction with DNA unless sigma70 is bound to the core component of RNA polymerase (RNAP). It is shown that interaction of sigma70 with the isolated beta' subunit of Escherichia coli RNAP is sufficient to induce unshielding of the DNA binding site. Using UV-induced DNA-protein cross-linking we demonstrate that free beta' stimulates specific cross-links between region 2 of the sigma70 polypeptide and a fragment of the non-template promoter strand containing the TATAAT sequence. Thus the sigmabeta' subassembly of RNAP can assume a functionally competent conformation independently of the bulk of the RNAP core.     

Conformational flexibility of σ70 in anti‐terminator loading

In promoter DNA, the preferred distance of the ?10 and ?35 elements for interacting with RNA polymerase‐bound σ⁷⁰ is 17 bp. However, the Devi et al. paper in this issue of Molecular Microbiology demonstrates that when the C‐terminal domain of σ⁷⁰, including the 3.2 linker, is not attached to the core enzyme, distances between 0 and 3 bp can be accommodated. This attests to the great flexibility of the 3.2 linker. The particularly stable complex with the 2 bp separation may lend itself to structural studies of an early elongation complex containing σ⁷⁰.