Highlights d A thermodynamic model quantitatively predicts PUM1/2 binding to any RNA sequence d Factors beyond simple recognition of consecutive residues influence binding d Comparison to X-linking data reveals thermodynamic control of PUM2 binding in cells d Analysis of RNA structure effects suggests disruption of RNA structure in cells
Highlights d Structure-guided design of PARP inhibitors d Identification of a potent and selective PARP11 inhibitor (ITK7) d ITK7 inhibits PARP11 auto-MARylation in cells d ITK7 causes PARP11 to dissociate from the nuclear envelope
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–λPR 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 Å. 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.
FRET (fluorescence
resonance energy transfer) between far-upstream
(−100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed
extensive bending and wrapping of λPR 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 (I1E), mid (I1M), and late (I1L)], followed
by conversion of {CC} to OC via I1L. FRET and PIFE are
first observed for I1E, not RPc. FRET and PIFE
together reveal large-scale bending and wrapping of upstream and downstream
DNA as RPC advances to I1E, 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 I1E. Converting I1E to I1M (∼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 I1M to I1L, 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, I1L 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.
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