Jeff Gelles

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.

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 (refs 4, 5). 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 pm s−1) to observe single molecules of a kinesin derivative moving slowly (∼2.5 nm s−1) at very low (150 nM) 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 (ref.7). Step distances other than 8 nm 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 8 nm coupling inferred from behaviour at low ATP can be generalized to high ATP.

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.

Ordered and Dynamic Assembly of Single Spliceosomes

Fluorescently labeled yeast spliceosome proteins reveal the events of intron splicing as it happens. The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

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.

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.

Forward and Reverse Motion of Single RecBCD Molecules on DNA

RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.

χ-Sequence recognition and DNA translocation by single RecBCD helicase/nucleasemolecules

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 ‘χ’ (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 ∼30 nm at 2 Hz) and to examine the mechanism by which χ 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 χ is negligible, showing that the activity modification at χ does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.

Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging

See How They Grow Controlled assembly and disassembly of the actin cytoskeleton is essential for processes such as cell motility, cytokinesis, and tumor metastasis. The formation of new actin filaments appears to involve the protein formin paired with another actin assembly-promoting factor. Breitsprecher et al. (p. 1164) used triplecolor single-molecule fluorescence microscopy to visualize actin assembly promoted by the formin, mDia1, and the tumor-suppressor, adenomatous polyposis coli (APC). The two assembly factors interacted directly to initiate filament assembly, after which mDia1 moved with the growing barbed ends while APC remained at the site of nucleation. Triple-color microscopy suggests that two factors interact to initiate actin formation and then separate as the filament grows. Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single-molecule fluorescence microscopy to image the tumor suppressor adenomatous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers initiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly and then separate but retain independent associations with either end of the growing filament.