Single-molecule studies of RNA polymerase: motoring along

Abstract

Single-molecule techniques have advanced our understanding of transcription by RNA polymerase (RNAP). A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps (OTs) have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNAP identifies a promoter, initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement the knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNAP, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

Figure 1

Atomic force microscopy. TECs are deposited onto an atomically flat surface (lower right panel). A microfabricated cantilever with a sharp tip is scanned over the sample. Deflections of the cantilever are registered by means of laser light reflected onto a position-sensitive detector (left panel). Detector signals are used to reconstruct a two-dimensional image (simulated image shown in the upper right panel)

Figure 2

Single-molecule fluorescence methods. Fluorescence may be used to track binding and residence times of accessory factors (upper panel) or the position of the RNAP holoenzyme by covalently attaching a fluorescent dye (yellow star) and exciting it with an appropriate wavelength (wavy blue arrows). FRET (lower panel) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. In the lower left panel, the donor (yellow) is excited (blue arrows) and emits light. When the donor fluorophore moves sufficiently close to the acceptor (lower right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.

Figure 3

Bead-based, single-molecule transcription assays. DNA is shown in blue, RNA in red, beads in light blue, optical traps in pink, fluorescent beads in yellow, magnets and magnetic beads in orange/blue. Directions of applied forces are shown by straight black arrows. The coverglass surface is indicated by a blue horizontal line. A) TPM assay. This method tracks transcriptional progress by averaging the Brownian excursions of a bead tethered by a changing length of DNA to a molecule of RNAP immobilized on the coverglass surface. B) Surface-based DNA-pulling OT assay. RNA polymerase is bound to a bead, and the distal end the DNA template is anchored to the coverglass. Force is exerted on the bead by an optical trap. Here, the force is shown assisting polymerase motion; reversal of the template direction allows the application of hindering loads. C) Surface-based RNA-pulling OT assay. A molecular handle comprised of dsDNA with a complementary ssDNA overhang is annealed to the 5′ end of the nascent RNA. As in (B), the RNAP is bound to a bead, and the DNA is anchored to the coverglass. Forces applied to the bead produce tension on the transcript. D) Pipette-based DNA-pulling assay. An RNAP molecule is bound to a bead held by a suction micropipette, and the distal end of the DNA template is attached to a second, free bead. Fluid flow exerts viscous forces on the free bead (right), placing tension on the tether. E) Dumbbell OT assay. Two beads, one attached to an RNAP molecule and the other to the distal end of the DNA template, are levitated above the surface by twin optical traps. Transcriptional progress of RNAP can be measured free of drift caused by motion of the coverglass surface. F) Fluorescent particle rotation assay. A larger bead is decorated with smaller fluorescent reporter beads, which can be used to determine its angle about a vertical axis. Similar to (A), the larger bead is tethered to a molecule of RNAP on the coverglass surface through the template DNA. Rotations of RNAP around the DNA template axis during elongation or promoter search lead to rotations of the larger bead that can be directly visualized. G) Magnetic tweezers assay. A superparamagnetic bead is tethered to one end of a DNA molecule whose distal end is attached to the coverglass surface. External magnets are used to impart both twist and small amounts of tension to the DNA. Rotations of these magnets underwind the DNA and induce the formation of plectonemes. Melting of the transcription bubble during initiation adds positive twist to the template, removing plectonemes and causing a large change in the height of the tethered bead that can be measured directly.

Figure 4

Promoter search and initiation. RNAP holoenzyme (core polymerase in green; σ-factor in purple) is postulated to find promoter target sequences through two mechanisms. Intersegment transfer involves the polymerase loosely binding to a position on the DNA, then making bridging contacts to a second position on the DNA before transferring from one position to the other. The sliding mechanism involves the diffusion of weakly-bound RNAP along the DNA. Once a promoter is found, the holoenzyme binds tightly to the DNA and bends it, forming the closed promoter complex. With the help of σ-factor, a portion of the DNA is melted, exposing bases of the template strand and forming the OPC. Subsequently, during abortive initiation, RNAP repeatedly synthesizes short RNA fragments before eventually clearing the promoter to form a stable TEC, and σ-factor is likely released.

Figure 5

Abortive initiation models. The ITC is shown with RNA polymerase in green, DNA in blue, RNA in red, and the enzyme active-site in yellow. Three mechanisms have been proposed to explain abortive initiation. In the transient excursions model (top), RNAP briefly breaks its contacts with the promoter region (horizontal green arrows) and transcribes a short segment of RNA. Upon release of the aborted product, RNAP diffuses back to restart the cycle. In the inchworming model (middle), flexible elements within the enzyme allow the footprint of RNAP to grow as RNA is synthesized, and promoter contacts at the upstream face are maintained (vertical green arrow). Upon release of the abortive RNA, the polymerase relaxes to its normal footprint. In the scrunching model (bottom), RNAP maintains its shape while increasing its effective footprint by pulling in some of the downstream DNA. Abortive loss of the RNA transcript then results in the release of this scrunched DNA, resetting the enzyme.

Figure 6

Transcription elongation pathway and a subset of off-pathway states. RNA polymerase is shown in green, the template strand in light blue, the non-template strand in dark blue, RNA in red, and ρ-factor in yellow. Elongation (central panel; green background) corresponds to the template-directed condensation of NTPs onto the 3′ end of the growing RNA chain. Individual nucleotides may occasionally be excised via pyrophosphorolysis, releasing NMPs and PP_i. A number of paused states branch off the central elongation pathway (upper panel; yellow background). An elemental pause state has been proposed as a common intermediate state preceding hairpin-stabilized and backtracking pauses (solid arrows), although both these states might be reached directly from the main elongation pathway (dotted arrows). Misincorporation-induced pauses are triggered when a mismatched NTP (yellow) is added to the RNA chain; backtracking often results in such cases. Two paths lead to transcriptional termination (lower panel; red background). Intrinsic termination occurs when RNAP transcribes specific sequence elements that code for a termination hairpin in the RNA followed by a U-rich tract, triggering dissociation of the TEC. Another termination pathway to termination involves the binding of ρ, which is thought to move along the transcript until reaching RNAP and ultimately dislodge the RNA from the enzyme.