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 energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes showed extensive bending/wrapping of {lambda}PR promoter DNA on E. coli RNA polymerase (RNAP) in closed and open complexes (CC, OC). Here we determine the kinetics and mechanism of DNA bending/wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye fluorescence enhancements (PIFE). FRET/PIFE kinetics exhibit two phases: rapidly-reversible steps forming a CC ensemble ({CC}c of four intermediates (initial (RPC), early (I1E), mid-(I1M), late (I1L)), followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET/PIFE together reveal large-scale bending/wrapping of upstream and downstream DNA as RPC advances to I1E, reducing -100/+14 distance to [~]75[A] 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 I1E. Converting I1E to I1M (~1s time-scale) reduces FRET efficiency with little change in -100/+14PIFE, interpreted as clamp-opening that moves far-upstream DNA (on {beta}) away from downstream DNA (on {beta}) to increase the -100/+14 distance by ~14[A]. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp-closing to reduce the -100/+14 distance by ~21[A]. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L converts 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/932780v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1068460org.highwire.dtl.DTLVardef@dcd6d9org.highwire.dtl.DTLVardef@9d4727org.highwire.dtl.DTLVardef@1aa8e47_HPS_FORMAT_FIGEXP M_FIG C_FIG