Closing and opening of the RNA polymerase trigger loop

Significance During transcription elongation at saturating nucleotide concentrations, RNA polymerase (RNAP) performs ∼50 nucleotide-addition cycles every second. The RNAP active center contains a structural element, termed the trigger loop (TL), that has been suggested, but not previously shown, to open to allow a nucleotide to enter and then to close to hold the nucleotide in each nucleotide-addition cycle. Here, using single-molecule fluorescence spectroscopy to monitor distances between a probe incorporated into the TL and a probe incorporated elsewhere in the transcription elongation complex, we show that TL closing and opening occur in solution, define time scales and functional roles of TL closing and opening, and, most crucially, demonstrate that one cycle of TL closing and opening occurs in each nucleotide-addition cycle.

Transcriptional activities of labelled RNAP derivatives were determined using fluorescence-detected transcription assays as described in (10) and are expressed relative to transcriptional activity of unlabelled E. coli RNAP measured under identical conditions. σ 70 E. coli σ 70 was prepared as in (11).
Streptolydigin (Stl) was the kind gift of Dr. E. Steinbrecher (Upjohn-Pharmacia, Kalamazoo, MI), and Salinamide A (Sal A) was the kind gift of Dr. W. Fenical (The Scripps Research Institute, La Jolla, CA). Rif was purchased from Sigma-Aldrich, and CBR703 was purchased from Maybridge. MccJ25 was prepared as in (13), and IX214A was prepared as in (14).
smFRET using confocal-ALEX Confocal-ALEX experiments were performed essentially as described (2,12). A green laser (532 nm; Compass 215M-20; Coherent) was used for direct excitation of the donor, and a red laser (638 nm; Radius 635-25; Coherent,) was used for direct excitation of the acceptor (2). Lasers were operated at continuous-wave excitation intensities of 120 μW at 532 nm and 80 μW at 638 nm and were alternated at 25 μs intervals using an acousto-optical modulator (Neos Technologies, Inc.). Fibercoupled collimated beams were directed to an Olympus IX71 inverted microscope (Olympus America, Inc.), reflected by a beam splitter, and focused into the sample through a 60x oil-immersion objective. Fluorescence emission from the sample was collected through the objective, filtered through a 100 μm pinhole, spectrally split by a dichroic mirror, and focussed onto two avalanche photodiode detectors (APD; SPCM-AQR-15; Perkin-Elmer).
Photons detected at the donor-emission channel upon donor excitation (F DD ), acceptor-emission channel upon donor excitation (F DA ), and acceptor-emission channel upon acceptor excitation (F AA ) were extracted based on photon arrival times. The stoichiometry parameter (S) was calculated for each above-threshold photon burst, as follows (2,12,15): The donor-acceptor smFRET efficiency (E*) for each above-threshold, photon burst was calculated as follows (2,12,15): Two-dimensional E*-S plots were used to distinguish species containing donor only (D-only), acceptor only (A-only), and both donor and acceptor (D-A). For species containing both donor and acceptor (D-A), one-dimensional E* histograms were plotted and were fitted with Gaussian curves (Figs. S4 and S7). The resulting histograms provided equilibrium population distributions of E*.
For experiments in Figs. 1C, 2, 3A-C, S6, and S9 in the presence of NTPs or NTP analogs, observation chambers containing immobilized fluorescent-probe-labelled, hexahistidine-tagged TECs (prepared as described above) were supplemented to the specified final concentrations with 30 μl solutions of NTPs or NTP analogs in imaging buffer, reaction mixtures were incubated 3 min at 22°C, and data were collected.
For experiments in Figs. 4 and S10, observation chambers containing immobilized fluorescent-probelabelled, hexahistidine-tagged TECs were prepared as described above with 30 μl imaging buffer in the observation chambers, data acquisition was started, and observation chambers were supplemented with 10μl of 20μM ATP in imaging buffer (yielding a final ATP concentration of 5 μM).
smFRET using TIRF-ALEX: data collection and data analysis smFRET experiments were performed using a custom-built objective-type total-internal-reflection fluorescence (TIRF) microscope (16). Light from a green laser (532 nm; Samba; Cobolt) and a red laser (635 nm; CUBE 635-30E, Coherent) was combined using a dichroic mirror coupled into a fiberoptic cable focused onto the rear focal plane of a 100x oil-immersion objective (numerical aperture 1.4; Olympus) and was displaced off the optical axis, such that the incident angle at the oil-glass interface of a stage-mounted observation chamber exceeded the critical angle, thereby creating an exponentially decaying evanescent wave (17). Alternating-laser excitation (ALEX; 12) was implemented by directly modulating the green and red lasers using an acousto-optical modulator (1205C, Isomet).
Fluorescence emission was collected from the objective, was separated from excitation light using a dichroic mirror (545 nm/650 nm, Semrock) and emission filters (545 nm LP, Chroma; and 633/25 nm notch filter, Semrock), was focused on a slit to crop the image, and then was spectrally separated (using a dichroic mirror; 630 nm DLRP, Omega) into donor and emission channels focused side-byside onto an electron-multiplying charge-coupled device camera (EMCCD; iXon 897; Andor Technology). A motorized x/y-scanning stage with continuous reflective-interface feedback focus (MS-2000; ASI) was used to control the sample position relative to the objective.
All data acquisition was carried out at 22°C. For all TIRF-ALEX experiments except those in Fig. 3D, laser powers were 4 mW (532 nm laser) and 0.75 mW (635 nm laser), and data were collected for 20 s using a frame rate of 1 frame per 20 ms. For experiments in Fig. 3D, laser powers were 1 mW (532 nm laser) and 0.3 mW (635 nm laser), and data were collected for 50 s using a frame rate of 1 frame per 100 ms. Fluorescence-emission intensities in donor-emission (green) and acceptor-emission (red) channels were detected using the peak-finding algorithm of the MATLAB (MathWorks) software package Twotone-ALEX, as described (16). Peaks detected in both emission channels (i.e., peaks for molecules containing both donor and acceptor probes) were fitted with two-dimensional Gaussian functions to extract background-corrected intensity-vs.-time trajectories for donor-emission intensity upon donor excitation (I DD ), acceptor-emission intensity upon donor excitation (I DA ), and acceptoremission intensity upon acceptor excitation (I AA ), as described (16). Intensity-vs.-time trajectories were curated to exclude trajectories exhibiting I DD <100 or >1,000 counts or I AA <200 or >1,000 counts, trajectories exhibiting multiple-step donor or acceptor photobleaching, trajectories exhibiting donor or acceptor photobleaching in frames 1-20, trajectories exhibiting donor or acceptor photoblinking, trajectories exhibiting E* values < 0.3 (inferred to be donor-only complexes or improperly assembled complexes), and portions of trajectories following donor or acceptor photobleaching.
Intensity-vs.-time trajectories were used to calculate trajectories of apparent donor-acceptor smFRET efficiency (E*) and donor-acceptor stoichiometry (S), as described (12,15): E*-vs.-S plots were prepared, S values were used to distinguish species containing only donor, only acceptor, and both donor and acceptor, and E* histograms were prepared for species containing both donor and acceptor, as described (12,15), and fitted to Gaussian distributions in Origin (Origin Lab). The resulting histograms provide equilibrium population distributions of E* states and, for each E* state, define mean E* (Figs. 1C, 3A, S5, S6 and S9B; gray bars and inset). E*-vs-time trajectories that, on visual inspection, exhibited transitions between distinct E* states and exhibited anti-correlated changes in DD (donor excitation-donor emission) and DA (donor excitationacceptor emission) channels were identified. For experiments in Figs. 1C, S5 and S9, few traces (<3%) showed dynamic behavior. For experiments in Figs. 2, S6 and S9, ~10% to ~60% of traces showed dynamic behavior. Dynamic E*-vs-time trajectories were analyzed globally to identify E* states by use of Hidden Markov Modelling (HMM) as implemented the Matlab (MathWorks) software package ebFRET (18), essentially as described (3,18). E*-vs-time trajectories were fitted to a two-state HMM model, E*-values from the fitted model were extracted, were plotted using Origin (Origin Lab), and were fitted to Gaussian distributions using Origin (Figs. 2B, 3B and S6; colored curves). The resulting histograms provide equilibrium population distributions of E* states and, for each E* state, define mean E* (Figs. 2B, 3B and S6; colored bars and inset). E* values were corrected, and accurate donor-acceptor efficiencies (E a ) and donor-acceptor distances (R) were calculated (Table S1) as described previously (4). From the accurate FRET efficiencies (E a ), distances (R; Table S1) were estimated using: where R 0 is the Főrster parameter for the donor/acceptor FRET pair [52 Å for DL550-DL650 FRET pair (19); 50 Å for DL550-Alexa647 FRET pair; calculated as: R 0 = 9780(n -4 κ 2 Q D J) 1/6 Å, where n is the refractive index of the medium, κ 2 is the orientation factor relating donor and acceptor transition dipoles (approximated as 2/3, noting that all mean E values are <0.5; 20), Q D is the donor quantum yield, and J is the overlap integral of donor emission and acceptor excitation].
Dwell times for E* states were extracted from HMM fits to E*-vs-time trajectories and were binned and plotted as distribution histograms in Origin (Fig. S5) For experiments in Figs. 4, S9, and S10, the open-TL dwell-time-distribution histograms were fit to a single-exponential function, and the closed dwell-time-distribution histograms were fit to a biexponential function with most events corresponding to short dwells (~60 ms; ~90%) and some events corresponding to longer dwells (~400 ms; ~10%). The rate of TL opening (k open ) and the rate of TL closing (k close ) were estimated from exponential fits to closed and open dwell time distribution histograms, respectively, as described above (Fig. S9D and S10).
The on-rate for ATP binding, the off-rate for ATP unbinding and the equilibrium dissociation constant for ATP binding were estimated from the TL-closing and TL-opening rates (assuming TL closing events occur upon NTP-binding events, and TL-opening events occur upon NTP-unbinding events), as follows (Fig. 2E, 3C, S9D Figs. 4 and S10, ~25% of molecules exhibited transitions between distinct E* states and exhibited anti-correlated changes in the DD (donor excitation-donor emission) and DA (donor excitation-acceptor emission) channels, upon addition of 5 μM ATP. One TL-closing step (transition from open-TL state to closed-TL state) through one TL-opening step (transition from closed-TL to open-TL state) was defined to constitute a TL closing-opening event. For each experiment in Fig. 4 (template directing addition of 1A, 2A, 3A, or 4A), a manual counting of numbers of TL closing-opening events was performed, and numbers of TL closing-opening events were plotted as a histogram in Origin. Rare complexes (<1%) that showed TL closing-opening events prior to ATP addition were excluded from the analysis. TL-closing and TL-opening rates were estimated as described above (Fig. S9). Molecules which did not show any transitions may include time traces where all transitions are missed or molecules which were unresponsive to addition of 5 μM ATP. Fig. S1. Use of smFRET to detect and characterize TL closing and opening in solution (A) Open-TL (left subpanel) and closed-TL (right subpanel) conformational states as observed in crystal structures of E. coli RNAP (23,24; PDB 5BYH and PDB 4YLN). Pink ribbon, species-specific sequence insertion 3 (SI3) in open-TL state. Light green ribbon, SI3 in closed-TL state. , pink. Other colors as in Fig. 1A. (B) Measurement of smFRET between first fluorescent probe incorporated at β' residue 942 in tip of E. coli RNAP TL (red sphere for open-TL state; green sphere for closed-TL) and second fluorescent probe incorporated at template-strand position +12 of downstream DNA (pink sphere). Inter-residue distances are ~41 Å for open-TL state and ~46 Å for closed-TL state. Other colors as in A.