Robert Landick

Stretching DNA with optical tweezers.

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.

Direct observation of base-pair stepping by RNA polymerase

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 sub-saturating 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.

Transcription Against an Applied Force

The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.

Structural basis for substrate loading in bacterial RNA polymerase

Water is predicted to be among, if not the most abundant molecular species after hydrogen in the atmospheres of close-in extrasolar giant planets (hot-Jupiters) Several attempts have been made to detect water on an exoplanet, but have failed to find compelling evidence for it or led to claims that should be taken with caution. Here we report an analysis of recent observations of the hot-Jupiter HD189733b taken during the transit, where the planet passed in front of its parent star. We find that absorption by water vapour is the most likely cause of the wavelength-dependent variations in the effective radius of the planet at the infrared wavelengths 3.6, 5.8 and 8 microns. The larger effective radius observed at visible wavelengths may be due to either star variability or the presence of clouds/hazes. We explain the most recent thermal infrared observations of the planet during secondary transit behind the star, reporting a non-detection of water on HD189733b, as being a consequence of the nearly isothermal vertical profile of the planet.s atmosphere. Our results show that water is detectable on extrasolar planets using the primary transit technique and that the infrared should be a better wavelength region than the visible, for such searches.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-Å resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5′-[(α,β)-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 α-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.

Backtracking by single RNA polymerase molecules observed at near-base-pair resolution

Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. 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.

Ubiquitous Transcriptional Pausing Is Independent of RNA Polymerase Backtracking

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.

The regulatory roles and mechanism of transcriptional pausing

The multisubunit RNAPs (RNA polymerases) found in all cellular life forms are remarkably conserved in fundamental structure, in mechanism and in their susceptibility to sequence-dependent pausing during transcription of DNA in the absence of elongation regulators. Recent studies of both prokaryotic and eukaryotic transcription have yielded an increasing appreciation of the extent to which gene regulation is accomplished during the elongation phase of transcription. Transcriptional pausing is a fundamental enzymatic mechanism that underlies many of these regulatory schemes. In some cases, pausing functions by halting RNAP for times or at positions required for regulatory interactions. In other cases, pauses function by making RNAP susceptible to premature termination of transcription unless the enzyme is modified by elongation regulators that programme efficient gene expression. Pausing appears to occur by a two-tiered mechanism in which an initial rearrangement of the enzymes active site interrupts active elongation and puts RNAP in an elemental pause state from which additional rearrangements or regulator interactions can create long-lived pauses. Recent findings from biochemical and single-molecule transcription experiments, coupled with the invaluable availability of RNAP crystal structures, have produced attractive hypotheses to explain the fundamental mechanism of pausing.

Bacterial Transcription Terminators: The RNA 3′-End Chronicles

The process of transcription termination is essential to proper expression of bacterial genes and, in many cases, to the regulation of bacterial gene expression. Two types of bacterial transcriptional terminators are known to control gene expression. Intrinsic terminators dissociate transcription complexes without the assistance of auxiliary factors. Rho-dependent terminators are sites of dissociation mediated by an RNA helicase called Rho. Despite decades of study, the molecular mechanisms of both intrinsic and Rho-dependent termination remain uncertain in key details. Most knowledge is based on the study of a small number of model terminators. The extent of sequence diversity among functional terminators and the extent of mechanistic variation as a function of sequence diversity are largely unknown. In this review, we consider the current state of knowledge about bacterial termination mechanisms and the relationship between terminator sequence and steps in the termination mechanism.

Regulator trafficking on bacterial transcription units in vivo.

The trafficking patterns of the bacterial regulators of transcript elongation sigma(70), rho, NusA, and NusG on genes in vivo and the explanation for promoter-proximal peaks of RNA polymerase (RNAP) are unknown. Genome-wide, E. coli ChIP-chip revealed distinct association patterns of regulators as RNAP transcribes away from promoters (rho first, then NusA, then NusG). However, the interactions of elongating complexes with these regulators did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from sigma(70) peaks in the direction of transcription and co-occurred with NusA and rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of rho did not increase RNAP levels within genes downstream from the RNAP peaks, suggesting the peaks are caused by a mechanism other than rho-dependent attenuation.

RNA Polymerase as a Molecular Motor

The existing single-molecule studies of E. coli RNAP suggest a number of promising avenues for future research into transcription mechanisms in both prokaryotes and eukaryotes. First, if measurements can be made precisely enough to resolve 1 bp steps with high time resolution, the experiments could discriminate between alternative translocation mechanisms and reveal essential features of RNAP chemomechanical coupling. In particular, such studies could differentiate between Brownian ratchet and power stroke translocation mechanisms since these models make quantitatively different predictions about the way that translocation step durations vary with applied force. Visualizing RNAP movements with single–base pair precision would also settle the question of whether >1 bp sliding movements are characteristic features of the ordinary chain elongation cycle. Second, single-molecule approaches are ideally suited to examining the differences in reaction kinetics between TECs in the same population that are following different, parallel reaction pathways (Erie et al. 1993xErie, D.A, Hajiseyedjavadi, O, Young, M.C, and von Hippel, P.H. Science. 1993; 262: 867–873CrossRef | PubMedSee all ReferencesErie et al. 1993) or that have heterogeneous structures. Third, single-molecule methods can not only detect movement of RNAP along the DNA, but also directly visualize RNAP attachment to and release from DNA. Therefore, the techniques can visualize transcription initiation and termination, making them powerful tools to investigate mechanisms of transcription regulation and the control of gene expression. Fourth, these techniques can visualize the large-scale structural changes in DNA or chromatin associated with transcription or transcription regulation. These include DNA looping (e.g.,3xFinzi, L and Gelles, J. Science. 1995; 267: 378–380CrossRef | PubMedSee all References, 12xRippe, K, Guthold, M, von Hippel, P.H, and Bustamante, C. J. Mol. Biol. 1997; 270: 125–138CrossRef | PubMed | Scopus (122)See all References), DNA bending (e.g.,Rippe et al. 1997xRippe, K, Guthold, M, von Hippel, P.H, and Bustamante, C. J. Mol. Biol. 1997; 270: 125–138CrossRef | PubMed | Scopus (122)See all ReferencesRippe et al. 1997), and chromatin rearrangements (seeFritzsche et al. 1995xFritzsche, W, Vesenka, J, and Henderson, E. Scanning Microsc. 1995; 9: 729–737PubMedSee all ReferencesFritzsche et al. 1995references therein). Single-molecule techniques can potentially give a more detailed structural picture than biochemical (e.g., gel-shift) methodologies and can observe changes in structure with high (in some cases, millisecond) time resolution.Perhaps the most important future applications of single-molecule microscopy techniques are analyses of biochemical pathways that involve assembly of large macromolecular complexes. Transcription and transcription regulatory systems involve the assembly of complex structures consisting of multiple protein molecules that interact with each other and with sites on the DNA and RNA. Despite the importance of these systems, in few cases has it been possible to define fully the kinetic mechanism of assembly (that is, the complete pathway of assembly and the rates of all steps) and its temporal relationship to catalytic and regulatory events. Recently, technology has been developed to visualize by fluorescence microscopy single protein molecules tagged with small organic dyes or expressed as fusions with green fluorescent protein (Funatsu et al. 1995xFunatsu, T, Harada, Y, Tokunaga, M, Saito, K, and Yanagida, T. Nature. 1995; 374: 555–559CrossRef | PubMedSee all ReferencesFunatsu et al. 1995). This should permit observation of the assembly of multiple proteins (perhaps each tagged with a different color fluorophore) into single transcription or regulatory complexes while translocation is simultaneously observed using one of the techniques described above. The technology is thus particularly well-suited to ask questions about the relationship between elongation factors binding to transcription complexes and their effects on the rate and persistence of transcription, because both the factor binding and kinetic changes can be individually monitored. For example, such studies could answer fundamental questions about the mechanisms of antitermination in bacteria, and in eukaryotes could reveal the temporal relationships between transcription complex assembly, regulatory factor binding, promoter escape, and conversion to an elongation-proficient TEC. By using polarization optics, single-molecule fluorescence microscopy can also directly observe reorientation of single dye molecules relative to the excitation light (Sase et al. 1997xSase, I, Miyata, H, Ishiwata, S, and Kinosita, K Jr. Proc. Natl. Acad. Sci. USA. 1997; 94: 5646–5650CrossRef | PubMed | Scopus (138)See all ReferencesSase et al. 1997). It thus has the potential to detect not only binding events but also structural reorganizations within single macromolecular complexes.Analysis of RNAPs as molecular motors is still in its infancy. Single-molecule experiments should improve our knowledge of the fundamental mechanisms of transcription and its regulation, particularly as the techniques become more widely used and better instrumentation is developed. These methods should be of value to study other DNA-based motor enzymes, including DNA polymerases, exo- and endonucleases, helicases, and topoisomerases.