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 have shown that it is the AT-rich Pminor spacer sequence, rather than promoter recognition elements or downstream DNA, that determines the effect of region 1.1 on promoter activity at Pminor. Substituting the Pminor spacer with a more GC-rich spacer or with the complement of the Pminor sequence results in a promoter that is equally active using polymerase with or without region 1.1. Furthermore, we find that the presence of the Pminor spacer, the GC-rich spacer, or the complement spacer results in different mobilities of promoter DNA during gel electrophoresis, suggesting that the spacer regions impart differing conformations or curvatures to the DNA. We speculate that the spacer can influence the trajectory of DNA as it enters the RNAP channel and that region 1.1 acts as a gatekeeper to monitor channel entry.
| 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 |
| Hook-Barnard, India G; Hinton, Deborah M (2007) Transcription initiation by mix and match elements: flexibility for polymerase binding to bacterial promoters. Gene Regul Syst Bio 1:275-93 |
| Hook-Barnard, India; Johnson, Xanthia B; Hinton, Deborah M (2006) Escherichia coli RNA polymerase recognition of a sigma70-dependent promoter requiring a -35 DNA element and an extended -10 TGn motif. J Bacteriol 188:8352-9 |
