Opening and closing of the bacterial RNA polymerase clamp

Abstract

Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

Fig. 1. Determination of RNAP clamp conformation in solution

(A) Measurement of smFRET between fluorescent probes incorporated at the tips of the RNAP β’ pincer (clamp) and the RNAP β pincer. Open (red), partly closed (yellow), and closed (green) RNAP clamp conformational states are as observed in crystal structures (PDB 1I3Q, 1HQM, and 1I6H). σ and the β' non-conserved domain are omitted for clarity in this and subsequent figures. (B) Incorporation of fluorescent probes at the tips of the RNAP β’ pincer (clamp) and the RNAP β pincer, by unnatural amino acid mutagenesis to incorporate 4-azidophenylalanine at sites of interest in β’ and β, followed by Staudinger ligation to incorporate fluorescent probes at 4-azidophenylalanines in β’ and β, followed by in vitro reconstitution of RNAP from labelled β’ and β and unlabelled α* (covalently linked α-N-terminal-domain dimer) and ω(see Supplemental Methods). Plasmids, genes, and proteins are shown as ovals, open bars, and closed bars, respectively. (C) Relationship between smFRET efficiencies, E, and RNAP clamp conformational states (see Supplemental Methods). The red boxes denote the open (model 4; red), closed (model 11; green), and collapsed (model 15; blue) clamp states observed in this work for RNAP-σ⁷⁰ holoenzyme (Fig. 2).

Fig. 2. RNAP clamp conformation in σ⁷⁰-dependent transcription initiation and elongation

Panels show histograms and Gaussian fits of observed donor-acceptor smFRET efficiencies, E (at left); mean E, mean R, and percentage, P, for each subpopulation (in inset at left); inferred structural states of the RNAP clamp (at right; colored as in Fig. 1C); and inferred extents of closure of the RNAP clamp (at far right; in degrees ±SEM relative to the open state). The vertical red lines denote mean E of the open, closed, and collapsed states in RNAP-σ⁷⁰ holoenzyme. (A) RNAP clamp conformation in RNAP holoenzyme, RPo, RPitc (4 nt RNA), and RDe (14 nt RNA). (B) Control three-color FRET experiments with third probe on σ⁷⁰ (data filtered to include only molecules containing σ⁷⁰). (C) Control three-color FRET experiments with third probe on DNA (data for RPo filtered to include only molecules containing DNA). (D) RNAP clamp conformation in RNAP core.

Fig. 3. RNAP clamp conformation in σ⁵⁴-dependent transcription initiation

(A) Intermediates in σ⁵⁴-dependent transcription initiation (23). (B) RNAP clamp conformation in RNAP-σ⁵⁴ holoenzyme, RPc, RPc+NtrC1, RPi1, RPi2, and RPo.

Fig. 4. Effects of inhibitors on RNAP clamp conformation

(A) Effects of inhibitors that block the isomerization of RPc to RPo: Myx, Cor, Rip, and Gp2. (B) Effects of inhibitors that block steps subsequent to the isomerization of RPc to RPo: Rif, Stl, and CBR. (C) Summary. The major clamp conformation at each step in transcription initiation and transcription elongation stage is shown in black. Minor clamp conformations are shown in gray. Myx, Cor, Rip, and Gp2, which inhibit the isomerization of RPc to RPo (vertical red line), inhibit clamp opening (slanted red lines); Rif, Stl, and CBR, which inhibit steps subsequent to the isomerization of RPc to RPo (vertical blue lines), do not inhibit clamp opening.