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

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.

Role of bacterial RNA polymerase gate opening dynamics in DNA loading and antibiotics inhibition elucidated by quasi-Markov State Model

“…In the later stage of the promoter melting, the loaded dsDNA will be separated into single-stranded DNA, and this step may require clamp opening. Furthermore, we also note that the structure of the promoter dsDNA may be more complicated than the DNA model used here, where the upstream promoter DNA (position −100) was found to wrap around the E. coli RNAP during promoter melting (71,72). In this situation, opening of the clamp was required even at the beginning of promoter melting.…”

Structural basis of ribosomal RNA transcription regulation

“…Mechanism of rRNA-specific transcription inhibition by DksA/ppGpp. Structural and biochemical studies of bacterial RNAP transcription suggest that the order of DNA loading around the TSS and DNA opening may be interchangeable during promoter recognition (i.e., DNA melts first outside RNAP (melt-load) or DNA melts after loading inside the RNAP cleft (load-melt)) depending on σ factors, promoters, transcription factors and conditions 15 , 33 , 41 , 42 . By combining structural and biochemical data from this and previous studies, we propose two pathways of RPo formation (Fig.…”

“…However, the rpsTP2 promoter for expressing ribosomal protein S20 is distinct from the rrnBP1 promoter that it contains G + C-rich DNA upstream of the −35 element and the TSS 7 bases downstream from the −10 element; therefore, it does not reveal the pathway for rRNA promoter complex formation and the mechanism of rRNA transcription regulation. In addition, the presence of TraR does not allow to infer the unperturbed pathway of the open complex formation by RNAP [15][16][17] . Here, we used cryo-EM to visualize the RNAP and rrnBP1 complexes and two additional complexes with DksA/ ppGpp on the way to RPo formation.…”

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

“…The holo docks on the promoter via concerted interactions with specific promoter regions known as UP (from around -60 to -40 region of the promoter relative to transcription start site at +1 position), -35, spacer, and -10 elements (8,(18)(19)(20)(21)(22). This initial unstable RNAP-promoter complex (RPC, where no DNA melting has occurred) isomerizes to more stable forms when the upstream and downstream regions of the promoter bend along the RNAP surface and into the DNA binding cleft, respectively (23)(24)(25)(26). The formation of catalytically active holo-promoter open complex (RPO) is completed when the -11/+2 region of the promoter DNA duplex unwinds and the template DNA strand enters the active site cleft of the RNAP (27)(28)(29)(30).…”

“…Most studies report the existence of two (32)(33)(34) or more (35) open complex structures (RPO, intermediates), but also several closed complex intermediates have recently been identified (26). In most cases, less than half of apparent the RPO's appear capable of productive promoter escape followed by full-length RNA synthesis (32,(36)(37)(38)(39).…”