Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F-x relationship for individual DNA molecules as short as -1 micron with unprecedented accuracy, subjected to both low (approximately 0.1 pN) and high (approximately 50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, Lp, of approximately 47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine 3+, reduced Lp to approximately 40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce Lp significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of approximately 1100 pN.

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) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.

The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated. Here we report the 3.0-A resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5'-[(alpha,beta)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin. In the EC/AMPcPP structure, the substrate binds to the active ('insertion') site closed through refolding of the trigger loop (TL) into two alpha-helices. In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive ('preinsertion') substrate configuration stabilized by streptolydigin-induced displacement of the TL. Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications. First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics. Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors.