Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by Escherichia coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

Abstract

FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λP_R promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RP_C), early (I_1E), mid (I_1M), and late (I_1L)], followed by conversion of {CC} to OC via I_1L. FRET and PIFE are first observed for I_1E, not RP_c. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RP_C advances to I_1E, decreasing the Cy3-Cy5 distance to ∼75 Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper β'-clamp while downstream DNA contacts the top of the β-pincer in I_1E. Converting I_1E to I_1M (∼1 s time scale) reduces FRET efficiency with little change in -100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on β') away from downstream DNA (on β) to increase the Cy3-Cy5 distance by ∼14 Å. FRET increases greatly in converting I_1M to I_1L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3-Cy5 distance by ∼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I_1L is converted to the initial unstable OC (I₂). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.