Transcription against an applied force

Science

Volumn 270, Issue 5242, 1995, Pages 1653-1657

  Yin, Hong ^a   Wang, Michelle D ^b   Svoboda, Karel ^c   Landick, Robert ^d   Block, Steven M ^b   Gelles, Jeff ^a  

^a BRANDEIS UNIVERSITY   (United States)

^b PRINCETON UNIVERSITY   (United States)

^c LUCENT TECHNOLOGIES   (United States)

^d UNIVERSITY OF WISCONSIN MADISON   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

KINESIN; MYOSIN; NUCLEOSIDE TRIPHOSPHATE; POLYSTYRENE; RNA POLYMERASE;

ARTICLE; CONTROLLED STUDY; CYTOSKELETON; DNA SEQUENCE; ENERGY METABOLISM; ESCHERICHIA COLI; GENETIC ANALYSIS; INTERFEROMETRY; NONHUMAN; PRIORITY JOURNAL; RNA SEQUENCE; RNA SYNTHESIS;

EID: 0029416931     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.270.5242.1653     Document Type: Article

References (42)

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4. M. J. Chamberlin, in The Harvey Lectures (Wiley-Liss, New York, 1994), Ser. 88, pp. 1-21.
5. P. R. Cook, Bioessays 16, 425 (1994).
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12. Sample preparation methods, buffers, and reaction conditions were the same as those used previously (7), except that disodium pyrophosphate (Sigma) was added at the specified concentration together with the NTPs, and 0.52-μm-diameter carboxylated polystyrene beads (Polysciences, Warrington, PA) were used in place of 0.226-μm beads. DNA template no. 5 (6), which contains the strong T7 A1 promoter followed by 3908 downstream base pairs, was used in all experiments. Transcription complex surface density was measured and controlled as in (7), so that each bead had a probability ≤0.20 of being attached to more than one transcription complex.
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14. _, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
15. tc has a time-varying component, due to the changing length of the DNA tether.
16. tc has a time-varying component, due to the changing length of the DNA tether.
17. trap) using free beads, either by measuring the power spectrum of the Brownian motion or by measuring the bead displacement from the trap center produced by viscous drag forces (9, 11, 12). Stiffnesses obtained with the use of these two methods differed by <10% for excursions below half the usable detector range (<100 nm). For larger excursions, the drag force method was used exclusively. Calibration was accomplished in two stages. First, the relation between digitized interferometer output voltage and true displacement (in nanometers) was established as described previously (9). Second, the relation between trapping force (in piconewtons) and displacement (in nanometers) was determined by the drag force method, with viscous forces being corrected for the proximity of the cover glass surface (12). The two data sets were then combined. In contrast to the case of silica particles (11), the force-displacement relation for polystyrene beads has significant nonlinearities in the outermost part of the trap; therefore, calibration data out to 150 nm were fit by a fifth-order polynomial to extrapolate interferometer signals into the region between 150 nm and ∼200 nm. This procedure may miscalculate the actual force produced in the region beyond 150 nm; however, exclusion from the analysis of data taken in this region did not significantly alter the mean stall forces reported here. The estimated force at 200 nm displacement was taken to be the largest measurable force at a given laser power.
18. tc) has not been applied to the bead velocity measurement. Therefore, this bead velocity represents a lower bound on the polymerase elongation rate.
19. -1 continuously for ≥5 s was taken to represent a stall. By this criterion, 13 of 77 complexes spontaneously underwent brief transient stalls that were apparently uncorrelated with the amount of force and were excluded from subsequent analyses. Such events may correspond to transcriptional pausing (1, 2) because movement resumed without the trap being repositioned.
20. Initially, 87 elongating complexes were observed at high laser powers. However, in 10 of them the bead spontaneously detached from its tether before the trap could be repositioned. The site of breakage could not be determined. Data from such complexes were excluded from further analysis. Eleven of the remaining 77 complexes did not stall before reaching the limit of the usable range of the trap.
21. D. A. Erie, O. Hajiseyedjavadi, M. C. Young, P. H. von Hippel, Science 262, 867 (1993).
22. Although inactive enzyme species induced by chemical stalling in solution can be reactivated by GreB protein [S. Borukhov, V. Sagitov, A. Goldfarb, Cell 72, 459 (1993)], the fraction of complexes that were irreversibly stalled by mechanical load did not decrease when 100 nM GreB protein was included in the buffer (24), which suggests either that these complexes are in a different state than that produced in the chemical stalling experiments or that GreB cannot act on complexes adsorbed to cover slips.
23. trap but increases the laser intensity at the RNA polymerase because the trap is moved closer to the enzyme molecule. Control experiments [at laser powers 82 to 99 mW; all reported laser powers are estimates in the specimen plane determined by the method of (11)] in which the trap was repositioned before the stall (in 14 of 15 complexes) subjected the transcription complexes to higher average light intensities and lower average trap forces than those in the stalling experiments. In the controls, complexes were inactivated in 82 ± 58 s (mean ± SD, n = 15) exposure to the laser, whereas in the stalling experiments first stalls (reversible or irreversible) occurred significantly earlier, in 38 ± 16 s (mean ± SD, n = 66), confirming that the first stalls cannot be explained by photodamage alone.
24. B. Krummel and M. J. Chamberlin, J. Mol. Biol. 225, 221 (1992).
25. stall. We estimated the magnitude of this effect at 26% or less, based on experiments in which beads fixed to a cover glass were moved in the specimen plane through the detector at various heights relative to the laser beam waist (24). Yet other uncertainties arise because the trap stiffness, hence the restoring force, varies with height, the trap being weaker in the vertical direction than in the horizontal direction. Given these various opposing effects, it seems reasonable to estimate the overall systematic errors in force at ≤∼30%.
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30. H. Yin et al., data not shown.
31. This estimate is a lower limit because it includes data from complexes that did not stall before reaching the trap limit and because we cannot exclude the possibility that some events classified as stalls may in fact be lengthy transcriptional pauses (16).
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38. stall. Although little such consumption has been reported for transcription complexes in solution [for example, see M. Chamberlin, R. L. Baldwin, P. Berg, J. Mol. Biol. 7, 334 (1963)), we cannot exclude the possibility that such uncoupled reactions are catalyzed by immobilized enzyme molecules subjected to the high applied forces used in the stalling experiments. Estimates of energy conversion efficiency from the stall forces of single kinesin and myosin molecules carry analogous uncertainties. Recent evidence (11) suggests that kinesin is not tightly coupled near stall.
39. s = 3 and 6 pN, respectively, in the two cited cases. However, this calculation may underestimate the force by a factor of ∼2 because supercoils in vivo are thought to be confined to a limited portion of the plasmid DNA.
40. s = 3 and 6 pN, respectively, in the two cited cases. However, this calculation may underestimate the force by a factor of ∼2 because supercoils in vivo are thought to be confined to a limited portion of the plasmid DNA.
41. L. F. Liu and J. C. Wang, Proc. Natl. Acad. Sci. U.S.A. 84, 7024 (1987).
42. Supported by grants from the National Institute of General Medical Sciences to J.G., R.L., and S.M.B. M.D.W. was supported by a Damon Runyon-Walter Winchell Cancer Research Fund postdoctoral fellow-ship. K.S. and S.M.B. thank the Rowland Institute for Science for support during the early stages of this work. A movie of the experiment shown in Fig. 2A can be viewed on the World Wide Web at http:// www.rose.brandeis.edu/users/gelles/stall/.