H-084. σ70 Region 1.1 of E. coli RNA Polymerase Modulates Transcription Initiation in Response to the AT-Richness of the Promoter Spacer

I. G. Hook-Barnard, D. M. Hinton;
NIH, Bethesda, MD.

Transcription initiation is a multistep process relying not only on recognition of promoter DNA, but also requiring structural isomerization of the entire RNAP/ promoter complex to form a machine competent for transcription. Much of this process is dependent upon the sigma subunit of the RNA polymerase holoenzyme. σ70 regions 2.4, 3.0 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 RNAP 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 RNAP active site. σ70 Region 1.1 plays a crucial role in the transient intermediate steps between RPc and RPo. However, Region 1.1, the N-terminal 100 amino acids of σ70, does not contact DNA, rather it appears to monitor isomerization of RNAP. FRET analysis and biochemical data from other labs indicate 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. Region 1.1 is essential for transition to RPo at the λPr promoter, and the movement of Region 1.1 may be coupled to late folding of the polymerase jaws, facilitating isomerization of RNAP into a stable (competitor resistant) RPo. Upstream DNA influences the efficiency of isomerization, possibly by contributing to the movement of Region 1.1. Furthermore, movement of Region 1.1 may modulate contact between Region 1.2 and the position -5 nucleotide,. Here, we investigate the role of Region 1.1 using RNAP reconstituted with either full-length sigma or sigma lacking Region 1.1. We verify our previous findings that the effect of Region 1.1 on transcription is not always positive, but rather varies with promoter. We find that the effect Region 1.1 has on promoter activity is not determined by core promoter elements (-35, TGn, -10). Instead, using derivatives of the Pminor promoter, we show that the influence of Region 1.1 can be altered by mutation of the promoter’s spacer sequence.