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.     

Predicting the strength of UP-elements and full-length E. coli σE promoters

Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σ(E), an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σ(E) regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths.