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. 2004 Apr 6;101(14):4776-80.
doi: 10.1073/pnas.0307241101. Epub 2004 Mar 22.

Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation

Andrey Revyakin  1 , Richard H Ebright, Terence R Strick
Affiliations

Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation

Andrey Revyakin et al. Proc Natl Acad Sci U S A. .

Abstract

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with approximately 1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximately 5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.

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Figures

Fig. 1.
Fig. 1.
Experimental approach. (A) Experimental setup. A double-stranded 4-kb DNA molecule containing a single promoter is tethered at one end, through multiple linkages, to a paramagnetic bead, and at the other end, through multiple linkages, to a glass surface. The DNA is torsionally constrained and mechanically stretched between the bead and the glass surface by application of a pair of magnets above the DNA helix axis. The distance between the bead and the surface, which reflects the DNA end-to-end extension (l), is monitored in real time by videomicroscopy. Upon rotation of the pair of magnets, the bead is rotated in lock-step register, superhelical turns are introduced into the DNA in lock-step register, plectonemic supercoils are formed, and, correspondingly, l is changed. (B) Calibration of l vs. number of superhelical turns. Over a broad range of positive and negative supercoiling, there is a linear relationship between l and the number of superhelical turns, with a change in l (δ) of 56 ± 5 nm per superhelical turn. (C and D) Detection of promoter unwinding. According to the relationship Lk = Tw + Wr (6), in a torsionally constrained DNA molecule with constant linking number (Lk), a change in twist (Tw; unwinding), must be compensated by an equal, but opposite, change in writhe (Wr; number of supercoils). With negatively supercoiled DNA, unwinding of approximately one turn of promoter DNA by RNAP must result in a compensatory loss of approximately one negative supercoil and, correspondingly, an increase in l (Δlobs,neg). With positively supercoiled DNA, unwinding of approximately one turn of promoter DNA by RNAP must result in a compensatory gain of approximately one positive supercoil and, correspondingly, a decrease in l (Δlobs,pos).
Fig. 2.
Fig. 2.
Detection of promoter unwinding. (A and B) Single-molecule traces of DNA extension vs. time for interaction of RNAP with a consensus promoter, as assessed with negatively supercoiled DNA (A; σ =–0.018; stable, effectively irreversible, promoter unwinding) and with positively supercoiled DNA (B; σ = 0.018; unstable, reversible promoter unwinding). Green points, raw data obtained at video rate (30 frames per s); red points, averaged data (1-s window); Δlobs,neg, transition amplitude with negatively supercoiled DNA; Δlobs,pos, transition amplitude with positively supercoiled DNA; Twait, time interval between a rewinding event and the next unwinding event; Tunwound, time interval between an unwinding event and the next rewinding event. (C) Histograms of Δlobs,neg (blue) and Δlobs,pos (red). The change in l attributable to DNA unwinding (Δlu) and the change in l attributable to DNA compaction arising from wrapping and/or bending (Δlc) are calculated as Δlu = (Δlobs,neg +Δlobs,pos)/2 and Δlc = (Δlobs,pos–Δlobs,neg)/2. The extent of unwinding (ΔTw) is calculated as ΔTw =Δlu/δ. (See Fig. 5; see also discussion in ref. .) (D–F) As in A–C, but with the rrnB P1 promoter (unstable, reversible promoter unwinding with negatively supercoiled DNA; no promoter unwinding with positively supercoiled DNA).
Fig. 3.
Fig. 3.
Kinetics and supercoiling-dependence of promoter unwinding. (A) Histograms of Twait and Tunwound (consensus promoter, positively supercoiled DNA; σ = 0.018; n = 1,127 events). (B) Tau plot (13) of values of Twait and Tunwound measured at a series of RNAP concentrations (consensus promoter, positively supercoiled DNA; n ≥ 100 events per RNAP concentration). KB, k2, and k–2, are, respectively, the equilibrium constant for formation of closed complex, the rate constant for unwinding, and the rate constant for rewinding in the standard kinetic scheme (refs. , , and ; see Eq. 1). The slope and y intercept of the tau plot of Twait yields (KBk2)–1 and (k2)–1; the y intercept of the tau plot of Tunwound yields (k–2)–1. (C) Supercoiling-dependence of Twait and Tunwound (consensus promoter, positively supercoiled DNA; σ = 0.008 to 0.024; n ≥ 100 events per σ value). The variable-torque and constant-torque regimes are defined based on the nonlinear and linear regimes of the experimental calibration curve in Fig. 1B (see ref. 18). Torque, |Γ|, is estimated to be 4.2, 4.4, 4.6, 5.0, and 5.0 pN nm for superhelical densities of 0.008, 0.011, 0.013, 0.019, and 0.024, respectively (refs. , , and and T.R.S., unpublished results). The magnitude of the supercoiling dependence in the variable-torque regime is consistent with a simple Arrhenius-law model in which torque biases the free energy of promoter unwinding/rewinding (see ref. 26).
Fig. 4.
Fig. 4.
Effects of ppGpp, effects of initiating nucleotide, and observation of promoter clearance. (A) Effects of ppGpp (0 or 100 μM) on stability of the unwound complex (consensus promoter, positively supercoiled DNA; σ = 0.018; n = 200 events). (B) Effects of ATP (0 or 2 mM) on stability of the unwound complex (consensus promoter, positively supercoiled DNA; σ = 0.018; n = 100 events). (C) Observation of promoter clearance by detection of successive, cumulative unwinding events, each corresponding to promoter clearance by RNAP molecule n, followed by promoter binding and unwinding by RNAP molecule n + 1 (consensus promoter, positively supercoiled DNA; 2 mM each NTP). (D–F) As in A–C, but for the rrnB P1 promoter, with negatively supercoiled DNA.
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