Transcription initiation is a multi-step process relying not only on recognition of promoter DNA, but also requiring structural isomerization of the entire RNA polymerase/promoter complex to form a machine competent for transcription. Much of this process is dependent upon the sigma subunit of the RNA polymerase. In E. coli the primary sigma is sigma70. Sigma70 regions 2.4, 3 and 4.2 contribute to double strand DNA recognition and binding via interactions with the -10, TGn, and -35 promoter elements, respectively. This initial binding of promoter DNA elements by RNA polymerase is termed the closed complex (RPc). For transcription to begin, the RPc must isomerize into the open complex (RPo), in which the DNA is single stranded and the template strand lies in the RNA polymerase active site. Sigma70 regions 2.3, 1.2, and 1.1 play crucial roles in the transient intermediate steps between RPc and RPo. Region 2.3 binds to single-stranded DNA in the -10 element, and region 1.2 contacts nucleotides at position -5. However, region 1.1, the N-terminal 100 amino acids of sigma70, does not contact DNA. Rather, it appears to monitor isomerization of RNA polymerase. Biophysical and biochemical analyses from other labs have indicated that the negatively charged region 1.1 lies within the RNAP channel during RPc. However, in RPo region 1.1 has been displaced by downstream DNA and is located outside the channel. At one promoter that has been studied, region 1.1 is essential for transition to RPo, and the movement of region 1.1 may be coupled to late folding of the polymerase jaws, facilitating isomerization of RNA polymerase into a stable (competitor resistant) RPo. Additionally, movement of region 1.1 could influence contact between region 1.2 and the position -5 nucleotide. Work in our lab has identified an unusual promoter, Pminor, whose initiation of transcription increases when sigma70 lacks region 1.1. Other tested promoters have been either unaffected or negatively affected by the lack of region 1.1. We asked what feature(s) of Pminor are associated with inhibition by Region 1.1. We have found, surprisingly, that it is the AT-rich spacer, rather than the sigma70-bound elements or the DNA downstream of the -10 element, that is responsible. Replacing the Pminor spacer sequence with its complement or a more GC-rich spacer generates a promoter unaffected by Region 1.1. In silica analyses predict different curvatures for these promoter DNAs. Furthermore, the presence of the Pminor spacer, the GC-rich spacer, or the complement spacer results in different mobilities of the DNA during electrophoresis, suggesting that the spacer imparts differing DNA conformations or curvatures. We speculate that the spacer influences the trajectory or flexibility of DNA as it enters the active site, and Region 1.1 acts as a gatekeeper to monitor this entry. We have also asked whether movement of Region 1.1 is a requirement for closing of the polymerase jaws onto the downstream DNA in RPo by comparing the nuclease protection footprints of RPo complexes of Pminor formed with polymerase with and without Region 1.1. We found that these footprints are quite similar with either polymerase and in both cases extend to +27, as expected for the RPo complex. Thus, at Pminor the polymerase jaws close sufficiently to protect the downstream DNA from nuclease cleavage, with or without the movement of Region 1.1. We conclude that the movement of Region 1.1 is not a prerequisite for jaw closure at all promoters.
| Hook-Barnard, India G; Hinton, Deborah M (2009) The promoter spacer influences transcription initiation via sigma70 region 1.1 of Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A 106:737-42 |
