Characterization of Transcription Initiation Intermediates in Escherichia Coli RNA Polymerase by Fluorescence

Abstract

Transcription initiation involves a series of conformational changes that are triggered in both RNA polymerase (RNAP) and DNA after recognition of −35, −10 elements of promoter DNA by RNAP holoenzyme, consisting of catalytic core and a specificity factor, σ. RNA polymerase (RNAP) functions as a molecular isomerization machine, binding and bending the promoter DNA in the initial closed complex and using binding free energy to melt 13 bp of DNA in the active site cleft to form an initial unstable open promoter complex, which is converted to a final open complex (RPo) (Saecker et al, '11). Promoter melting is thought to commence upon flipping of −11A of the non-template strand out of the DNA helix (Schroeder et al, '09) but several questions remain open. After recruitment of RNAP to promoter DNA, what conformational changes occur in the complex to put the downstream DNA duplex in the cleft? When and how is the DNA bent and wrapped around RNAP? When does the −11A element rotate vis-a-vis wrapping? To answer these questions, we are characterizing early intermediates at the λPr promoter using equilibrium and kinetic bulk fluorescence measurements. Fluorescence Resonance Energy Transfer (FRET) experiments monitor the extent of wrapping whereas stopped-flow “Beacon” assays provide information about −11A base-flipping and opening of −10 region (Mekler et al, '11). FRET experiments show that promoter DNA is wrapped around RNAP in the advanced closed complex and that wrapping persists in open complexes. The wrapped open complex is found to be transcriptionally competent. Real-time stopped-flow kinetic experiments are in progress to determine the sequence of steps in which upstream and downstream duplex promoter DNA are bent toward and wrapped around RNAP to put the start site region in the cleft and open it.