Bending and Wrapping of Upstream Promoter DNA on E. coli RNA Polymerase Facilitates Open Complex Formation in Transcription Initiation; A Fluorescence (FRET, PIFE) Study

Abstract

Removal of the far‐upstream region of promoter DNA reduces the isomerization rate of closed complexes (CC) to open complexes (OC) of Escherichia coli RNA polymerase (RNAP) and λPR DNA by 1–2 orders of magnitude. Upstream DNA is proposed to facilitate OC formation by wrapping on RNAP and remodeling the downstream cleft to allow bending of the downstream duplex into the cleft prior to opening. Here we report stopped flow fluorescence kinetic studies of open complex formation at λPR promoter DNA labeled at far‐upstream and downstream positions with Cy3 and/or Cy5 fluorescent probes. We determine the time course of forming the CC ensemble (10 ms – 3 s at 19 C), in which the upstream and downstream duplex DNA are bent and wrapped on RNAP. Cy3 to Cy5 FRET (fluorescence resonance energy transfer) detects wrapping, and single‐dye PIFE (protein‐induced fluorescence enhancement) detects transient contacts of the far‐upstream DNA with the downstream cleft, and contacts of the downstream DNA as it is bent into the cleft. Subsequently (3 s – 400 s) we determine the time course of the isomerization of the CC ensemble to OC, in which the upstream DNA remains bent and wrapped but where contacts of the upstream DNA with the downstream cleft are released. These results are consistent with the model for facilitation of isomerization by wrapping of upstream DNA, and with previous footprinting and kinetic information.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.