Single RNA Polymerase Molecules Research Articles

Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution.

During transcriptional elongation, RNA polymerases (RNAP) employ a stepping mechanism to translocate along the DNA template while synthesizing RNA. Optical trapping assays permit the progress of single molecules of RNA polymerase to be monitored in real time, at resolutions down to the level of individual base pairs. Additionally, optical trapping assays permit the application of exquisitely controlled, external forces on RNAP. Responses to such forces can reveal details of the load-dependent kinetics of transcriptional elongation and pausing. Traditionally, the bacterial form of RNAP from E. coli has served as a model for the study of transcriptional elongation using optical traps. However, it is now feasible to perform optical trapping experiments using the eukaryotic polymerase, RNAPII, as well. In this report, we describe the methods to perform optical trapping transcriptional elongation assays with both prokaryotic RNAP and eukaryotic RNAPII. We provide detailed instructions on how to reconstitute transcription elongation complexes, derivatize beads used in the assays, and perform optical trapping measurements.

Single-Molecule Analysis of Transcription-Coupled Repair

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER), has been known to lead to more efficient repair than the global NER repair (GGR). Here we use magnetic trapping of single DNA molecules to study the interactions of TCR proteins with stalled RNA polymerase (RNAP). Single RNAP molecules are stalled on a DNA transcript using either a CTP-less cassette or a thymine-thymine dimer located on the transcribed strand. Stalled RNAP is displaced by the Mfd translocase, and a long-lived intermediate is formed. We characterize the interaction between UvrA/UvrB proteins and the long-lived intermediate formed upon Mfd displacement of RNAP. This interaction leads to formation of a pre-incision complex that is catalytically competent for DNA incision, and the activity of this complex is tested for in the magnetic trap upon addition of UvrC.

Real-Time Initial Transcription by a Multisubunit RNA Polymerase

Abortive initiation, the repetitive synthesis of short RNA transcripts before promoter escape and elongation, is a highly dynamic process that can constitute the rate-limiting step for many gene promoters. Previous studies using single-molecule Förster Resonance Energy Transfer (smFRET) demonstrated that abortive initiation occurs via a DNA-scrunching mechanism mediated by RNA polymerase. While these studies, along with biochemical work, have yielded insight into abortive initiation, no single-molecule method has been able to directly capture RNA synthesis/release, or the associated conformational changes of the transcription complex. Here, we descibe a highly sensitive smFRET assay that, for the first time, monitors transcription bubble expansion and compaction during abortive RNA synthesis and release in real-time; we also performed similar analysis using an assay that reports on the movement of downstream DNA toward the RNA polymerase main channel. Using single-molecule time trajectories, we have observed multiple cycles (up to 50) of RNA synthesis by single RNA polymerase molecules and we used them to obtain the rates of RNA synthesis and release, as well as to study the dependence of the rates on nucleotide concentration and promoter sequence. Our initial results revealed that, surprisingly, the rate of nucleotide addition in initial transcription is 5-10 fold faster than elongation; moreover, we observed large heterogeneity between different RNA polymerase molecules with regards to specific steps in initial transcription; such heterogeneity may contribute to the noise associated with gene expression.

E. coli NusG Inhibits Backtracking and Accelerates Pause-Free Transcription by Promoting Forward Translocation of RNA Polymerase

NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of ribonucleoside triphosphate concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, that is, the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime (“stabilized”) pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state toward the post-translocated state.

Single-Molecule, Motion-Based DNA Sequencing Using RNA Polymerase

We present a method for sequencing DNA that relies on the motion of single RNA polymerase molecules. When a given nucleotide species limits the rate of transcription, polymerase molecules pause at positions corresponding to the rare base. An ultrastable optical trapping apparatus capable of base pair resolution was used to monitor transcription under limiting amounts of each of the four nucleotide species. From the aligned patterns of pauses recorded from as few as four molecules, we determined the DNA sequence. This proof of principle demonstrates that the motion of a processive nucleic acid enzyme may be used to extract sequence information directly from DNA.

Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules

Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.

Imaging RNA polymerase–amelogenin gene complexes with single molecule resolution using atomic force microscopy

The AMELX gene encoding the enamel matrix protein, amelogenin, is located within (and in the opposite orientation to) the first intron of the ARHGAP6 gene, which encodes a GTPase-activating protein. The orientation of these two genes with respect to each other raises the possibility that they may undergo simultaneous convergent transcription during amelogenesis. The aim of this study was to use atomic force microscopy (AFM) to study a transcriptionally active amelogenin DNA template and to investigate the binding of RNA polymerase to convergently aligned promoters. Images of RNA polymerases stalled on DNA templates were obtained following incubation of the template with RNA polymerases and ribonucleotide triphosphates. A linear DNA template incorporating an intact rat amelogenin cDNA flanked by convergently aligned coliphage T7 and T3 promoters was constructed and shown to be transcriptionally active in vitro. Atomic force microscopy images of transcription complexes revealed globular structures, corresponding to single RNA polymerase molecules bound at specific locations on the DNA templates. These results indicate that AFM allows the visualization of individual RNA polymerases on DNA templates, offering a realistic approach to investigating the concept of convergent transcription of nested genes, which may lead to an understanding of whether the simultaneous expression of AMELX and ARHGAP6 is possible during the formation of tooth enamel.

Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules

Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.

Studies on biomolecules using single molecule imaging and manipulation techniques

The best way to obtain unambiguous information about the function of biomolecules is to study their function at the single molecule level. By attaching a small tag such as a single fluorescent dye molecule to biomolecules, or a tag that is huge compared to the size of a biomolecule, we have been able to image the individual behaviours in real time under an optical microscope. Huge tags such as micrometer-sized plastic beads also allow the manipulation of individual molecules with, e.g. optical or magnetic tweezers. Using these techniques, we have observed individual behaviours of RNA polymerase, a molecular machine that copies the genetic information on DNA onto a messenger RNA. We found that some of the RNA polymerase molecules underwent linear diffusion along DNA, helping RNA polymerase to search for the promoter. We also found that a single RNA polymerase molecule rotated around the right-handed screw axis of the double helix of DNA during transcription with a rotary torque of >5 pN nm. The present methods are potentially applicable to the examination of a wide variety of protein–nucleic acid interactions, especially those involved in the process of transcription.

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 ( approximately 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.

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase.

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Nonequilibrium mechanism of transcription termination from observations of single RNA polymerase molecules.

Cessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized. To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized Escherichia coli RNA polymerase (RNAP) molecules in vitro. An RNAP molecule remains at a canonical intrinsic terminator for approximately 64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator. Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator. The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex.

Single-Molecule Imaging of RNA Polymerase-DNA Interactions in Real Time

Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of λ-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of ∼0.66 s −1, which was more than severalfold smaller than the rate at other positions. The association rate constant for the slow dissociation sites was 9.2 × 10 2 bp −1 M −1 s −1. The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions. RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA. The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA. The present method is potentially applicable to the examination of a wide variety of protein–nucleic acid interactions, especially those involved in the process of transcription.

Force and velocity measured for single molecules of RNA polymerase.

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.

Chapter 7 Single Molecule Imaging and Nanomanipulation of Biomolecules

This chapter discusses the single molecule imaging and nanomanipulation of biomolecules. The methods described in the chapter are applied to examining single nucleotidase reactions of other enzymes. It is possible to suspend a single DNA in solution by manipulation with dual optical traps, as well as to see single fluorescently labeled RNA polymerases. Directly observing the reading process of the DNA genetic information by a single RNA polymerase molecule is not just a dream now, but realistic. The techniques for single molecule imaging and manipulation is very powerful for studies not only of motility of motor proteins, but also of molecular genetics, signal transduction and processing in cell, dynamic molecular process of proteins.

Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule

Schafer et al. (Nature 352:444-448 (1991)) devised the tethered particle motion (TPM) method to detect directly the movement of single, isolated molecules of a processive nucleic acid polymerase along a template DNA molecule. In TPM studies, the polymerase molecule is immobilized on a glass surface, and a particle (e.g., a 0.23 microns diameter polystyrene bead) is attached to one end of the enzyme-bound DNA molecule. Time-resolved measurements of the DNA contour length between the particle and the immobilized enzyme (the "tether length") are made by determining the magnitude of the Brownian motion of the DNA-tethered particle using light microscopy and digital image processing. We report here improved sample preparation methods that permit TPM data collection on transcript elongation by the Escherichia coli RNA polymerase at rates (approximately 10(2)-fold higher than those previously obtained) sufficient for practical use of microscopic kinetics techniques to analyze polymerase reaction mechanisms. In earlier TPM experiments, calculation of tether length from the observed Brownian motion was based on an untested numerical simulation of tethered bead Brownian motion. Using the improved methods, we have now empirically validated the TPM technique for tether lengths of 308-1915 base pairs (bp) using calibration specimens containing particles tethered by individual DNA molecules of known lengths. TPM analysis of such specimens yielded a linear calibration curve relating observed Brownian motion to tether length and allowed determination of the accuracy of the technique and measurement of how temporal bandwidth, tether length, and other experimental variables affect measurement precision. Under a standard set of experimental conditions (0.23 microns diameter bead, 0.23 Hz bandwidth, 23 degrees), accuracy is 108 and 258 bp r.m.s. at tether lengths of 308 and 1915 bp, respectively. Precision improves linearly with decreasing tether length to an extrapolated instrumentation limit of 10 bp r.m.s. and improves proportionally to the inverse square root of measurement bandwidth (1.9 x 10(2) bp Hz-1/2 for 1090-bp tethers). Measurements on large numbers of individual polymerase molecules reveal that time-averaged single-molecule elongation rates are more variable than is predicted from the random error in TPM measurements, demonstrating that the surface-immobilized RNA polymerase molecules are kinetically heterogeneous.

Visualization of single molecules of RNA polymerase sliding along DNA.

Transcription requires that RNA polymerase binds to promoters buried in nonspecific sites on DNA. The search for promoters may be facilitated if the polymerase slides along the molecule of DNA. Single molecules of Escherichia coli RNA polymerase were visualized, and their movements on immobilized bacteriophage lambda and T7 DNAs were examined. Deviating from drifts by bulk flow, about 40 percent of the enzyme molecules moved along the extended DNA. The results provide direct evidence for sliding as a mechanism for relocation of the enzyme on DNA.

Transcription by single molecules of RNA polymerase observed by light microscopy

The kinetics of transcription by Escherichia coli RNA polymerase relate directly to the regulation of transcription and to the properties of processive enzymes in general, but analysis of RNA polymerase movement along the DNA template has so far been limited to the study of populations of enzyme molecules. The ability to view nanometre-sized particles with the light microscope suggested a method of monitoring transcription by individual RNA polymerase molecules. We describe here the behaviour of 40-nm-diameter particles of colloidal gold attached to the ends of DNA molecules being transcribed by RNA polymerase immobilized on a glass surface. The tethered gold particles are released from the surface at times after addition of nucleoside triphosphates that are consistent with the kinetics of transcription by RNA polymerase in solution. Analysis of the brownian motion of the gold particles enabled us to measure the movement along the template DNA of individual polymerase molecules.

Studies of the binding of Escherichia coli RNA polymerase to DNA : I. The role of sigma subunit in site selection

A membrane filter assay has been devised to study the binding of Escherichia coli RNA polymerase to DNA. RNA polymerase is quantitatively retained on nitrocellulose filters, and DNA bound in complexes between RNA polymerase and labeled T7 DNA is retained with an efficiency of up to 70%. The attachment of a single RNA polymerase molecule to a molecule of T7 DNA is sufficient to cause retention of the DNA molecule on the filter. Using the filter assay, it has been shown that both RNA polymerase holoenzyme and core polymerase bind to T7 DNA. The interaction of RNA polymerase holoenzyme with T7 DNA leads to two classes of complex. Approximately eight polymerase molecules can bind to each T7 DNA molecule to form a highly stable complex ( K assoc. = 10 12 to 10 14 m −1). In addition, there are many other sites on the DNA molecule at which a weak complex can be formed ( K assoc. = 10 8 to 10 8 m −1). The majority of holoenzyme molecules bound in the highly stable complex are thought to be bound at or near the T7 early promoter region, while the weak binding sites are thought to reflect a low, general affinity of holoenzyme for other regions of the DNA helix. In contrast, only a single major class of binding sites is found on T7 DNA for core polymerase ( K assoc. = 2 × 10 11 m −1) and there are many such sites on the T7 genome. These sites are interpreted as reflecting a high general affinity of core polymerase for all regions of the DNA helix. It is concluded that RNA polymerase can exist in two conformational states, one programmed for promoter binding and initiation (holoenzyme) and the other designed for RNA chain elongation (core polymerase). Sigma subunit may function in site selection primarily to switch the enzyme reversibly between these two states.