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

FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of lambda P-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 (RPC), early (I-1(E)), 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-1(E), not RPC. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I-1(E), decreasing the Cy3-Cy5 distance to similar to 75 angstrom and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper beta'-clamp while downstream DNA contacts the top of the beta-pincer in I-1(E). Converting I(1)(E )to I-1M (similar to 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 beta') away from downstream DNA (on beta) to increase the Cy3-Cy5 distance by similar to 14 angstrom. FRET increases greatly in converting IIh1 to 1 1 ,, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3-Cy5 distance by similar to 21 angstrom. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I-1L is converted to the initial unstable OC (I-2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.