E. Coli RNA Polymerase: A Molecular DNA Opening Machine

Abstract

Using “burst” kinetic methods to obtain near-homogeneous populations of transient intermediates for fast footprinting, together with solute probing experiments, we have characterized the series of large-scale conformational changes in E. coli RNA Polymerase (RNAP) and λPR promoter DNA that occur in “isomerization” of the initial recognition complex to form the stable open complex (RPo). We find that RNAP is a molecular isomerization machine that uses wrapping of upstream DNA to place the start-site region of the downstream duplex in the cleft (shown by HO protection and FRET in the closed intermediate I1).1 Then, in the rate determining step, RNAP opens the entire 13 bp initiation bubble (as judged by permanganate reactive thymines) and places the start site base of the template strand in the active site to form a transient open intermediate (I2).2 After this opening/ untwisting step, RNAP stabilizes the open DNA conformation by step-wise assembly of an encircling downstream jaw/ clamp, together with repositioning of the nontemplate strand in the cleft to form the more stable open intermediate I3 and convert it to the stable open complex RPo at λPR.2,3To test these conclusions, we are investigating deletions of the downstream “jaw” of RNAP [B’1149-1190], upstream and/ or downstream DNA, and/ or region 1.1 of σ70 subunit of RNAP [1-98] on the kinetics of formation and lifetime of open complexes. Remarkably, many of these deletions have large effects on the rate-determining, DNA-opening step, suggesting a synergy between upstream, downstream and in-cleft interactions in this sophisticated molecular machine.1. Davis et al. ’07; Drennan, Saecker et al., in preparation2. Gries et al. ‘103. Kontur, Gries et al.’06, ’08, ‘10Supported by NIH GM23467