Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules.

Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.

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.

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.

One-headed kinesin derivatives move by a nonprocessive, low-duty ratio mechanism unlike that of two-headed kinesin.

A single molecule of the "two-headed" motor enzyme kinesin can move along a microtubule continuously for many enzymatic turnovers (processive movement), and the velocity produced by one kinesin molecule is the same as that produced by many kinesin molecules (high duty ratio). We studied the microtubule movement driven at 1 mM ATP by biotinated N-terminal fragments of Drosophila kinesin heavy chain attached to streptavidin-coated coverslips at various surface densities. K448-BIO has velocity at a high density of vmax = 750 nm s-1 and is dimeric (hence two-headed); K365-BIO (vmax = 200 nm s-1) and K340-BIO (vmax = 90 nm s-1) are monomeric. Escape of microtubules from the surface was prevented by methylcellulose so that continuous trajectories of microtubules not continuously attached to motor molecules could be recorded by video microscopy. The component of instantaneous velocity parallel to the microtubule axis (v) was analyzed in trajectories with a mean velocity 0.4-0.7 times vmax. In K448-BIO trajectories, the distribution of v was bimodal with peaks near 0 and 750 nm s-1. Temporal autocorrelation analysis of v detected lengthy episodes of high-velocity movement consistent with isolated processive microtubule runs driven at vmax by single K448-BIO dimers. K365-BIO and K340-BIO trajectories had unimodal distributions of v and autocorrelation times much shorter than those for K448-BIO. Therefore the monomeric motors have duty ratio < 55% (i.e., no forward movement is generated for at least 45% of the enzymatic cycle time) or processivity below the detection limit of approximately 300 turnovers even in methylcellulose. Continuous movement at maximal velocity thus requires more than one kinesin head.

Release of isolated single kinesin molecules from microtubules.

Previous studies on the motor enzyme kinesin suggesting that the enzyme molecule tightly binds to a microtubule by only one of its two mechanochemical head domains were performed with multiple kinesin molecules on each microtubule, raising the possibility that interactions between adjacent bound molecules may interfere with the binding of the second head. To characterize the microtubule-bound state of isolated single kinesin molecules, we have measured the rates of nucleotide-induced dissociation of the complex between microtubules and bead-labeled single molecules of the dimeric kinesin derivative K448-BIO using novel single-molecule kinetic methods. Complex dissociation by <2 microM ADP displays an apparent second-order rate constant of 1.2 x 10(4) M-1 s-1. The data suggest that only one of the two heads is bound to the microtubule in the absence of ATP, that binding of a single ADP to the complex is sufficient to induce dissociation, and that even lengthy exposure of kinesin to the microtubule fails to produce significant amounts of a two-head-bound state under the conditions used. The inhibitor adenylyl imidodiphosphate (AMP-PNP) induces stochastic pauses in the movement of bead-labeled enzyme molecules in 1 mM ATP. Exit from pauses occurs at 2 s-1 independent of AMP-PNP concentration. The same rate constant is obtained for dissociation of the transient kinesin-microtubule complexes formed in 1 mM ADP, 0.5 mM AMP-PNP, suggesting that release of a single AMP-PNP molecule from the enzyme is the common rate-limiting step of the two processes. The results are consistent with alternating-sites movement mechanisms in which two-head-bound states do not occur in the enzyme catalytic cycle until after ATP binding.

Postoperative mitral valve abscess. A case report.

The authors report a case of a patient with postoperative perimitral bioprosthetic abscess and probable systemic embolization. The location of the abscess is not common and the use of porcine bioprostheses is supposedly associated with a low rate of embolic complications. Because of the high mortality and morbidity, it is stressed that patients with prosthetic valve endocarditis should be managed aggressively with surgery.

Coupling of kinesin steps to ATP hydrolysis.

A key goal in the study of the function of ATP-driven motor enzymes is to quantify the movement produced from consumption of one ATP molecule. Discrete displacements of the processive motor kinesin along a microtubule have been reported as 5 and/or 8 nm. However, analysis of nanometre-scale movements is hindered by superimposed brownian motion. Moreover, because kinesin is processive and turns over stochastically, some observed displacements must arise from summation of smaller movements that are too closely spaced in time to be resolved. To address both of these problems, we used light microscopy instrumentation with low positional drift (< 39 pms[-1]) to observe single molecules of a kinesin derivative moving slowly (approximately 2.5nm s[-1]) at very low (150nM) ATP concentration, so that ATP-induced displacements were widely spaced in time. This allowed increased time-averaging to suppress brownian noise (without application of external force), permitting objective measurement of the distribution of all observed displacement sizes. The distribution was analysed with a statistics-based method which explicitly takes into account the occurrence of unresolved movements, and determines both the underlying step size and the coupling of steps to ATP hydrolytic events. Our data support a fundamental enzymatic cycle for kinesin in which hydrolysis of a single ATP molecule is coupled to a step distance of the microtubule protofilament lattice spacing of 8.12 nm. Step distances other than 8nm are excluded, as is the coupling of each step to two or more consecutive ATP hydrolysis reactions with similar rates, or the coupling of two 8-nm steps to a single hydrolysis. The measured ratio of ATP consumption rate to stepping rate is invariant over a wide range of ATP concentration, suggesting that the 1 ATP to 8nm coupling inferred from behaviour at low ATP can be generalized to high ATP.

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.

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 and functional features of one- and two-headed biotinated kinesin derivatives.

The oligomeric structure was determined for four recombinant kinesin derivatives containing N-terminal fragments of the kinesin alpha-subunit. Some of the proteins were dimeric (two-headed) molecules with mechanochemical properties similar to those of intact kinesin. Comparison of the primary and quaternary structures of the derivatives with those of intact kinesin suggests that structures distinct from the long alpha-helical coiled-coil rod domain contribute to subunit self-association. Three of the proteins contain a single engineered site for post-translational biotination in vivo; this facilitates analysis of motility in experiments in which the proteins are specifically bound to streptavidin-conjugated microscopic plastic beads. One of the derivatives is monomeric (one-headed); like the two-headed derivatives, it is functional in the motility assay and is a microtubule-dependent ATPase. Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme fails to track microtubule protofilaments. This confirms a prediction of proposed "hand-over-hand" mechanisms of kinesin movement. The ability of molecules with a one-headed solution structure to generate movement is consistent with a translocation-generating conformational change internal to the kinesin head. A simple set of coupling rules can be used to formulate consistent mechano-chemical mechanisms that explain movement by both one- and two-headed kinesin molecules.

Failure of a single-headed kinesin to track parallel to microtubule protofilaments.

Kinesin, a two-headed motor enzyme molecule, hydrolyses ATP to direct organelle transport along microtubules. As it moves along a microtubule, kinesin remains associated with, or 'tracks', microtubule protofilaments. We have prepared truncated kinesin derivatives that contain either two mechanochemical head domains or only a single head. Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme frequently fails to track protofilaments, suggesting that it detaches from microtubules during movement. In this way, the one-headed kinesin derivative is similar to the motor enzyme myosin, which frequently detaches from the actin filament during movement. For myosin (which has two heads), the consequence of this detachment is that single molecules do not appear to drive continuous movement along the filament. Our observations suggest that the ability of single two-headed kinesin molecules to drive continuous movement results from a 'hand-over-hand' mechanism in which one head remains bound to the microtubule while the other detaches and moves forwards.

Subunit interactions in dimeric kinesin heavy chain derivatives that lack the kinesin rod.

The N-terminal residues of the two heavy chains of the motor enzyme kinesin form two globular "heads"; the heads are attached to a "rod" domain which is a two-stranded alpha-helical coiled-coil. Interaction between the heads is thought to be important to kinesin function. The rod may not be necessary for head-head interactions because a heavy chain N-terminal fragment containing only residues from the head and adjacent region forms dimers (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). However, the nature and stability of the subunit-subunit interactions in such derivatives are unclear. To examine the physical properties of heavy chain interaction in and near the head domains, we characterized the self-association behavior of two dimeric kinesin derivatives predicted (Lupas, A., van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) to lack the rod. Derivative K448-BIO contains the 448 N-terminal residues of Drosophila kinesin heavy chain fused at the C terminus to a 2-residue linker and a C-terminal fragment from Escherichia coli biotin carboxyl carrier protein; derivative K448-L is the same except that it lacks the biotin carboxyl carrier protein fragment. Both derivatives expressed in insect cells display microtubule-stimulated ATPase activity; K448-BIO also displays microtubule motility. Equilibrium sedimentation and gel filtration indicate that purified K448-BIO and K448-L at 0.02-0.4 mg/ml form homogeneous solutions of homodimers with no detectable formation of monomers or higher order oligomers. Derivative self-association is non-covalent but extremely stable with an association constant > or = 2 x 10(8) M-1. Stable subunit-subunit association induced by structures in and near the kinesin heads may be necessary for full mechanochemical function.

Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules.

In gene regulatory systems in which proteins bind to multiple sites on a DNA molecule, the characterization of chemical mechanisms and single-step reaction rates is difficult because many chemical species may exist simultaneously in a molecular ensemble. This problem was circumvented by detecting DNA looping by the lactose repressor protein of Escherichia coli in single DNA molecules. The looping was detected by monitoring the nanometer-scale Brownian motion of microscopic particles linked to the ends of individual DNA molecules. This allowed the determination of the rates of formation and breakdown of a protein-mediated DNA loop in vitro. The measurements reveal that mechanical strain stored in the loop does not substantially accelerate loop breakdown, and the measurements also show that subunit dissociation of tetrameric repressor is not the predominant loop breakdown pathway.

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.

Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface.

Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.

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.