The promoter spacer influences transcription initiation via σ ⁷⁰ region 1.1 of Escherichia coli RNA polymerase

Abstract

Transcription initiation is a dynamic process in which RNA polymerase (RNAP) and promoter DNA act as partners, changing in response to one another, to produce a polymerase/promoter open complex (RPo) competent for transcription. In Escherichia coli RNAP, region 1.1, the N-terminal 100 residues of 70 , is thought to occupy the channel that will hold the DNA downstream of the transcription start site; thus, region 1.1 must move from this channel as RPo is formed. Previous work has also shown that region 1.1 can modulate RPo formation depending on the promoter. For some promoters region 1.1 stimulates the formation of open complexes; at the P minor promoter, region 1.1 inhibits this formation. We demonstrate here that the AT-rich P minor spacer sequence, rather than promoter recognition elements or downstream DNA, determines the effect of region 1.1 on promoter activity. Using a P minor derivative that contains good 70 -dependent DNA elements, we find that the presence of a more GC-rich spacer or a spacer with the complement of the P minor sequence results in a promoter that is no longer inhibited by region 1.1. Furthermore, 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 or flexibility of DNA as it enters the RNAP channel and that region 1.1 acts as a ''gatekeeper'' to monitor channel entry.

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

“…The precise origin of these hypersensitivities is unclear, but previous results have indicated an effect of deletion of σ 1.1 on interactions with regions upstream of the active site cleft. DNase footprints with a variant RNAP lacking σ 1.1 at the promoter Pmin show upstream enhancements around −40 and −50 [37]. At some promoters, the effects of σ 1.1 deletion on the kinetics of OC formation and the structure of OC also depend on the length and sequence of the promoter spacer [37, 38].…”

Coupling of Downstream RNA Polymerase–Promoter Interactions with Formation of Catalytically Competent Transcription Initiation Complex

“…Although σ1.1 is not strictly required for in vitro transcription, it modulates the kinetics of RP o formation at some promoters (28,29). σ1.1 is also involved in inhibition of transcription initiation by T7 phage gp2 protein (30, 31).…”

Mutations That Stimulate flhDC Expression in Escherichia coli K-12

“…While there is no consensus sequence for spacers, studies of specific promoters with significantly altered spacer sequences and compositions have led to the proposal that RNAP senses overall spacer structure and that spacer flexibility is required to maintain contact with the promoter DNA during transcription initiation (64,(66)(67)(68)(69). How RNAP might sense spacer conformation is not clear.…”

“…There may be contact between the Zn 2ϩ -binding domain of the ␤= subunit and the distal portion of the spacer, as indicated from structural studies of Thermus aquaticus RNAP (70). Residue R451 in the linker between domains 2 and 3 of 70 interacts with spacer position Ϫ18 in a manner that is affected by local curvature (69), and 70 region 1.1, while not contacting DNA, affects the transition from the closed to the open complex in a way that is influenced by spacer structure (68).…”