1 research outputs found
Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open <i>Escherichia coli</i> RNA Polymerase−λP<sub>R</sub> Promoter Complexes
Initial
recognition of promoter DNA by RNA polymerase (RNAP) is
proposed to trigger a series of conformational changes beginning with
bending and wrapping of the 40–50 bp of DNA immediately upstream
of the −35 region. Kinetic studies demonstrated that the presence
of upstream DNA facilitates bending and entry of the downstream duplex
(to +20) into the active site cleft to form an advanced closed complex
(CC), prior to melting of ∼13 bp (−11 to +2), including
the transcription start site (+1). Atomic force microscopy and footprinting
revealed that the stable open complex (OC) is also highly wrapped
(−60 to +20). To test the proposed bent-wrapped model of duplex
DNA in an advanced RNAP−λP<sub>R</sub> CC and compare
wrapping in the CC and OC, we use fluorescence resonance energy transfer
(FRET) between cyanine dyes at far-upstream (−100) and downstream
(+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies
are observed for the CC (0.30 ± 0.07) and the OC (0.32 ±
0.11) for both probe orientations. Fluorescence enhancements at +14
are observed in the single-dye-labeled CC and OC. These results demonstrate
that upstream DNA is extensively wrapped and the start site region
is bent into the cleft in the advanced CC, reducing the distance between
positions −100 and +14 on promoter DNA from >300 to <100
Ã…. The proximity of upstream DNA to the downstream cleft in the
advanced CC is consistent with the proposed mechanism for facilitation
of OC formation by upstream DNA