During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 ± 0.6 Å, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and subsaturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.Processive molecular motors tend to move in discrete steps 1 . Recent advances in singlemolecule techniques have made it possible to observe such steps directly at length scales of a few nanometres or greater. The ability to detect individual catalytic turnovers, as monitored through motor displacement, while simultaneously controlling the force, substrate concentration, temperature or other parameters, provides a means to probe the mechanisms responsible for motility. Single-molecule measurements of stepping have supplied fresh insight into the mechanisms responsible for motion in motor proteins such as myosin, kinesin, dynein and the F 1 -ATPase 2-9 . A number of processive nucleic acid-based enzymes, such as lambda exonuclease 10,11 , RecBCD helicase 12-14 and RNAP 15-19 , have also been studied successfully by single-molecule methods, but the comparatively small size of their steps has been experimentally inaccessible up to this point. Movements through a single base pair along double-stranded DNA correspond to a displacement of just ~3.4 Å (ref. 20), which is more than 20-fold smaller than the 8-nm kinesin step 4 and sevenfold smaller than the 2-3-nm resolution limit attained in most previous work 2, 3,14,19,21 . During transcription, E. coli RNAP translocates along DNA while following its helical pitch 22 , adding ribonucleoside triphosphates (NTPs) successively to the growing RNA. The basic reaction cycle consists of binding the appropriate NTP, incorporation of the associated nucleoside monophosphate into the RNA, and release of pyrophosphate. In addition to † Present address: Department of Integrative Biology, University of California, Berkeley, California 94720, USA. * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. The mechanism that leads to translocation during transcriptional elongation continues to be debated 27-32 , and at least two classes of models have been proposed....

RNA polymerase (RNAP) transcribes DNA discontinuously, with periods of rapid nucleotide addition punctuated by frequent pauses. We investigated the mechanism of transcription by measuring the effect of both hindering and assisting forces on the translocation of single Escherichia coli transcription elongation complexes, using an optical trapping apparatus that allows for the detection of pauses as short as one second. We found that the vast majority of pauses are brief (1-6 s at 21 degrees C, 1 mM NTPs), and that the probability of pausing at any particular position on a DNA template is low and fairly constant. Neither the probability nor the duration of these ubiquitous pauses was affected by hindering or assisting loads, establishing that they do not result from the backtracking of RNAP along the DNA template. We propose instead that they are caused by a structural rearrangement within the enzyme.

Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo 1 . Its low error rate may be achieved by means of a 'proofreading' mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3′ end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events (~5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.Recent studies have implicated the nucleolytic activity of RNA polymerase as part of a proofreading mechanism 2-4 , similar to that found in DNA polymerases 5 . A key feature of this proofreading mechanism is a short backtracking motion of the enzyme along the DNA template (directed upstream, opposite to the normal direction of transcriptional elongation). Similar rearward movements are thought to accompany the processes of transcriptional pausing 6-8 , arrest 9,10 , and transcription-coupled DNA repair 11 . During backtracking, the transcription bubble shifts and the DNA-RNA hybrid duplex remains in register, while the 3′ end of the RNA transcript moves away from the active site, and may even protrude into the secondary channel (nucleotide entrance pore) of the enzyme 6,7,9 , blocking the arrival of ribonucleoside triphosphates (NTPs). In its backtracked state, RNAP is able to cleave off and discard the most recently added base(s) by endonucleolysis, generating a fresh 3′ end at the active site for subsequent polymerization onto the nascent RNA chain. In this fashion, short RNA segments carrying misincorporated bases can be replaced, leading to the correction of transcriptional errors (Fig. 1a). Accessory proteins have been identified that increase transcriptional fidelity by preferentially stimulating the cleavage of misincorporated nucleotides: GreA and GreB for E. coli RNA polymerase 4 and SII/TFIIS for eukaryotic RNA polymerase II 2,3 .Correspondence and requests for materials should be addressed to S.M.B. (sblock@stanford.edu).. * These authors contributed equally to this work Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. We studied transcription by RNAP at physiological nucleotide concentrations using a new single-molecule assay tog...

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.Virtually all RNA-processing and DNA-processing enzymes show selectivity for backbone compositions or base sequences of their nucleic-acid substrates. This substrate selectivity is especially crucial for the HIV-1 reverse transcriptase (RT), which binds and discriminates between a variety of nucleic-acid duplexes for distinct catalytic functions 1,2 . RT is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains 3,4 , catalysing a complex, multi-step reaction to convert the single-stranded RNA genome into double-stranded DNA 1,2 . First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA 11-13 , thereby creating the double-stranded DNA viral genome. Specific cleavage by RNase H then removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral DNA into the host chromosome 14 . Inappropriate initiation of synthesis of the plus-strand DNA at other RNA segments prevents integration 2,15 . RT must therefore obey the following primer-selection rules: first, DNA primers readily engage the polymerase activity of RT; second, generic RNA primers are not efficiently extended by RT but readily engage the RNase H activity of RT when annealed with DNA; third, the PPT RNA ca...

Optical traps are useful for studying the effects of forces on single molecules. Feedback-based force clamps are often used to maintain a constant load, but the response time of the feedback limits bandwidth and can introduce instability. We developed a novel force clamp that operates without feedback, taking advantage of the anharmonic region of the trapping potential where the differential stiffness vanishes. We demonstrate the utility of such a force clamp by measuring the unfolding of DNA hairpins and the effect of trap stiffness on opening distance and transition rates.Optical traps (also known as optical tweezers) use light from a tightly focused laser beam to trap small, polarizable objects, such as dielectric beads, in a three-dimensional potential well centered near the focal point [1]. For sufficiently small displacements from equilibrium, the trapping potential is harmonic: The restoring force F varies linearly with the displacement from the trap center x with a constant stiffness k that can be calibrated by several well-established methods. A number of groups have used optical traps in this linear regime to study the properties of important biological systems by attaching single molecules to microscopic beads. Such systems include motor proteins (e.g., kinesin [2], myosin [3], and dynein [4]), processive nucleic acid enzymes (e.g., polymerases [5,6], helicases [7], and exonucleases [8]), and nucleic acid structures [9,10].For biophysical experiments, a force clamp that fixes the load on the bead (a mechanical analog of the voltage clamp widely used in neuroscience) offers several advantages. The maintenance of constant load facilitates measurement of position by eliminating the need for series elastic corrections to displacement signals [11,12]. Furthermore, constant force avoids complications arising from changes to the potential energy landscape that are generated by molecular motions against a changing load. Techniques such as magnetic tweezers [13] and laminar fluid flow [14] can be used to generate constant force but have been limited in practice to no better thañ 10 nm / Hz spatial resolution and have, thus far, been unable to match the resolution attained by optical traps.In optical traps, a force clamp is typically implemented using a feedback system that measures the instantaneous position of the trapped object and then moves the trap to maintain a set displacement between the object and the trap center. Such active force clamps have proven to be powerful tools for biomechanical studies, but they suffer from two inherent limitations.