No abstract

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.

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the “open” promoter complex (OC), of synthesis of a short RNA–DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA–DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA–RNAP contacts and escape of RNAP from the promoter.

FRET (fluorescence energy transfer) between farupstream (-100) and downstream (+14) cyanine dyes showed extensive bending/wrapping of λPR 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 ~75Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper b'-clamp while downstream DNA contacts the top of the b-pincer in I1E. Converting I1E to I1M (~1s time-scale) reduces FRET efficiency with little change in -100/+14PIFE, interpreted as clampopening that moves far-upstream DNA (on b') away from downstream DNA (on b) to increase the -100/+14 distance by ~14Å. 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 ~21Å. 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.

In transcription initiation, specific contacts between RNA polymerase (RNAP) and promoter DNA are disrupted as the RNA-DNA hybrid advances into the cleft, resulting in escape of RNAP. From the pattern of large and small rate constants for steps of initiation at λPR promoter at 19°C, we proposed that in-cleft interactions are disrupted in extending 3-mer to 5-mer RNA, −10 interactions are disrupted in extending 6-mer to 9-mer, and −35 interactions are disrupted in extending 10-mer to 11-mer, allowing RNAP to escape. Here we test this mechanism and determine enthalpic and entropic activation barriers of all steps from kinetic measurements at 25°C and 37°C. Initiation at 37°C differs significantly from expectations based on lower-temperature results. At low concentration of the second iNTP (UTP), synthesis of full-length RNA at 37°C is slower than at 25°C and no transient short RNA intermediates are observed, indicating a UTP-dependent bottleneck step early in the 37°C mechanism. Analysis reveals that the 37°C λPR OC (RPO) cannot initiate and must change conformation to a less-stable initiation complex (IC) capable of binding the iNTP. We find that IC is the primary λPR OC species below 25°C, and therefore conclude that IC must be the I3 intermediate in RPO formation. Surprisingly, Arrhenius activation energy barriers to five steps where RNAP-promoter in-cleft and −10 contacts are disrupted are much smaller than for other steps, including a negative barrier for the last of these steps. We interpret these striking effects as enthalpically-favorable, entropically-unfavorable, stepwise bubble collapse accompanying disruption of RNAP contacts.SignificanceTranscription initiation is highly regulated. To understand regulation, mechanisms of initiation and escape of RNA polymerase (RNAP) from the promoter must be understood. RNAP forms a highly-stable open complex (RPO) with λPR promoter at 37°C. From experiments determining effects of temperature on rate constants for each step of RNA synthesis, we find that RPO cannot bind the initiating nucleotides, that the I3 intermediate and not RPO is the initiation complex, and that contacts of RNAP with single-stranded DNA of the discriminator and −10 region and with −35 duplex DNA are disrupted stepwise as the RNA-DNA hybrid moves into the cleft. Evidence is obtained for stepwise bubble collapse and base stacking accompanying disruption of interactions of the single-stranded discriminator and −10 regions with RNAP.