The 0 degree C closed complexes between Escherichia coli RNA polymerase and two promoters, T7-A3 and lacUV5

The promoter-specific binding of Escherichia coli RNA polymerase to the T7-A3 and the lacUV5 promoters at 0 degrees C was analyzed by DNase I footprinting. At 37 degrees C, the footprint from RNA polymerase bound to the A3 promoter is essentially the same as that reported by Galas, D.J., and Schmitz, A., (1978) Nucleic Acids Res. 5, 3157-3170 for the lacUV5 promoter. At 0 degrees C, the footprint for the A3 promoter is well defined but reduced in size. The principal difference between the 0 and 37 degrees C footprints is a region from -2 to +18 which is protected by polymerase at the higher but not at the lower temperature. In contrast, the 0 degree C footprint for the lacUV5 promoter differs substantially in character from the footprint for A3 at 0 degree C. The footprint is similar to the pattern of DNase I digestion of DNA bound to a surface; alternating regions of sensitive and protected DNA are spaced at intervals of about 10 base pairs. This region of DNase I-sensitive and -resistant DNA has the same boundaries as the 0 degree C footprint on T7-A3. Temperature shift experiments confirmed the sequence specificity of the RNA polymerase interaction with UV5 at 0 degree C. These results indicate that RNA polymerase binds specifically to each promoter sequence in a closed complex. The increased time and amounts of RNA polymerase required to form the 0 degree C footprint on the lacUV5 promoter indicate that it binds RNA polymerase more weakly than does the T7-A3 promoter. Therefore there is a correlation between the binding constant for closed complex formation estimated from kinetic measurements and the formation of the 0 degree C footprint. The -35 region of the promoter may be more important in establishing the 0 degree C footprint because the T7-A3 promoter is a better match to the consensus sequence. Conversely, the -10 region seems less important because lacUV5 is a perfect match to the consensus, whereas the T7-A3 promoter matches at only five out of seven positions. The 0 degree C footprints encompass both regions along with the spacer; the combination of these regions rather than an individual region may determine the character of the footprint and the magnitude of the binding constant.     

On the role of the Escherichia coli RNA polymerase sigma 70 region 4.2 and alpha-subunit C-terminal domains in promoter complex formation on the extended -10 galP1 promoter

Bacterial promoters of the extended -10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended -10 Escherichia coli galP1 promoter at temperatures as low as 6 degrees C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended -10 motif and/or containing the -35 promoter consensus element. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no alpha-subunit C-terminal domains (alpha CTD) and either wild-type sigma 70 subunit or sigma 70 lacking region 4.2 was assembled and tested for promoter complex formation. At 37 degrees C, alpha CTD and sigma 70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended -10 motif. However, no promoter complexes formed when both alpha CTD and sigma 70 region 4.2 were absent. Thus, in the context of an extended -10 promoter, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for sigma 70 region 4.2 but not alpha CTD was required for open promoter complex formation on galP1 derivatives with extended -10 motif. We propose a model involving sigma 70 region 4.2 interaction with the beta flap domain that explains these observations.