메뉴 건너뛰기




Volumn 282, Issue 5390, 1998, Pages 902-907

Force and velocity measured for single molecules of RNA polymerase

Author keywords

[No Author keywords available]

Indexed keywords

KINESIN; MYOSIN; RNA POLYMERASE;

EID: 0032582494     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5390.902     Document Type: Article
Times cited : (792)

References (43)
  • 1
    • 0027106595 scopus 로고
    • M. J. Chamberlin, Harvey Lect. 88, 1 (1994); S. Uptain, C. Kane, M. Chamberlin, Annu. Rev. Biochem. 66, 117 (1997); R. Mooney, I. Artsimovitch, R. Landick, J. Bacteriol. 180, 3265 (1998).
    • (1994) Harvey Lect. , vol.88 , pp. 1
    • Chamberlin, M.J.1
  • 2
    • 0031008221 scopus 로고    scopus 로고
    • M. J. Chamberlin, Harvey Lect. 88, 1 (1994); S. Uptain, C. Kane, M. Chamberlin, Annu. Rev. Biochem. 66, 117 (1997); R. Mooney, I. Artsimovitch, R. Landick, J. Bacteriol. 180, 3265 (1998).
    • (1997) Annu. Rev. Biochem. , vol.66 , pp. 117
    • Uptain, S.1    Kane, C.2    Chamberlin, M.3
  • 3
    • 0031801743 scopus 로고    scopus 로고
    • M. J. Chamberlin, Harvey Lect. 88, 1 (1994); S. Uptain, C. Kane, M. Chamberlin, Annu. Rev. Biochem. 66, 117 (1997); R. Mooney, I. Artsimovitch, R. Landick, J. Bacteriol. 180, 3265 (1998).
    • (1998) J. Bacteriol. , vol.180 , pp. 3265
    • Mooney, R.1    Artsimovitch, I.2    Landick, R.3
  • 4
    • 0029416931 scopus 로고
    • H. Yin et al., Science 270, 1653 (1995).
    • (1995) Science , vol.270 , pp. 1653
    • Yin, H.1
  • 6
    • 0028143652 scopus 로고
    • D. A. Schafer, J. Gelles, M. P. Sheetz, R. Landick, Nature 352, 444 (1991); H. Yin, R. Landick, J. Gelles, Biophys. J. 67, 2468 (1994).
    • (1994) Biophys. J. , vol.67 , pp. 2468
    • Yin, H.1    Landick, R.2    Gelles, J.3
  • 7
    • 0027749617 scopus 로고
    • H. Kabata et al., Science 262, 1561 (1993); S. Kasas et al., Biochemistry 36, 461 (1997).
    • (1993) Science , vol.262 , pp. 1561
    • Kabata, H.1
  • 8
    • 0031028425 scopus 로고    scopus 로고
    • H. Kabata et al., Science 262, 1561 (1993); S. Kasas et al., Biochemistry 36, 461 (1997).
    • (1997) Biochemistry , vol.36 , pp. 461
    • Kasas, S.1
  • 9
    • 0030969278 scopus 로고    scopus 로고
    • R. Landick, Cell 88, 741 (1997); J. Gelles and R. Landick, ibid. 93, 13 (1998).
    • (1997) Cell , vol.88 , pp. 741
    • Landick, R.1
  • 10
    • 0032478549 scopus 로고    scopus 로고
    • R. Landick, Cell 88, 741 (1997); J. Gelles and R. Landick, ibid. 93, 13 (1998).
    • (1998) Cell , vol.93 , pp. 13
    • Gelles, J.1    Landick, R.2
  • 12
    • 0028362896 scopus 로고
    • K. Svoboda and S. M. Block, Cell 77, 773 (1994); A. J. Hunt, F. Gittes, J. Howard, Biophys. J. 67, 766 (1994); C. M. Coppin, D. W. Pierce, L. Hsu, R. D. Vale, Proc. Natl. Acad. Sci. U.S.A. 94, 8539 (1997); H. Kojima, E. Muto, H. Higuchi, T. Yanagida, Biophys. J. 73, 2012 (1997).
    • (1994) Cell , vol.77 , pp. 773
    • Svoboda, K.1    Block, S.M.2
  • 13
    • 0028145209 scopus 로고
    • K. Svoboda and S. M. Block, Cell 77, 773 (1994); A. J. Hunt, F. Gittes, J. Howard, Biophys. J. 67, 766 (1994); C. M. Coppin, D. W. Pierce, L. Hsu, R. D. Vale, Proc. Natl. Acad. Sci. U.S.A. 94, 8539 (1997); H. Kojima, E. Muto, H. Higuchi, T. Yanagida, Biophys. J. 73, 2012 (1997).
    • (1994) Biophys. J. , vol.67 , pp. 766
    • Hunt, A.J.1    Gittes, F.2    Howard, J.3
  • 14
    • 0030786388 scopus 로고    scopus 로고
    • K. Svoboda and S. M. Block, Cell 77, 773 (1994); A. J. Hunt, F. Gittes, J. Howard, Biophys. J. 67, 766 (1994); C. M. Coppin, D. W. Pierce, L. Hsu, R. D. Vale, Proc. Natl. Acad. Sci. U.S.A. 94, 8539 (1997); H. Kojima, E. Muto, H. Higuchi, T. Yanagida, Biophys. J. 73, 2012 (1997).
    • (1997) Proc. Natl. Acad. Sci. U.S.A. , vol.94 , pp. 8539
    • Coppin, C.M.1    Pierce, D.W.2    Hsu, L.3    Vale, R.D.4
  • 15
    • 0030824684 scopus 로고    scopus 로고
    • K. Svoboda and S. M. Block, Cell 77, 773 (1994); A. J. Hunt, F. Gittes, J. Howard, Biophys. J. 67, 766 (1994); C. M. Coppin, D. W. Pierce, L. Hsu, R. D. Vale, Proc. Natl. Acad. Sci. U.S.A. 94, 8539 (1997); H. Kojima, E. Muto, H. Higuchi, T. Yanagida, Biophys. J. 73, 2012 (1997).
    • (1997) Biophys. J. , vol.73 , pp. 2012
    • Kojima, H.1    Muto, E.2    Higuchi, H.3    Yanagida, T.4
  • 18
    • 3643097081 scopus 로고    scopus 로고
    • note
    • Transcription complexes were prepared as described previously and attached to 0.5-μm-diameter polystyrene beads (2, 3). Voltage signals corresponding to displacement in the interferometer and laser light intensity were low-pass filtered at 1 kHz, digitally sampled by computer at 2 kHz, then averaged and binned at 20 Hz for offline analysis. The interferometer was calibrated and signals were corrected as previously described (9). The corrections adjust for the changes in interferometer sensitivity as beads move vertically away from the trap center and for the geometry as the angle of the DNA changes with respect to the plane of the coverglass during shortening of the tether.
  • 19
    • 3643071025 scopus 로고    scopus 로고
    • note
    • Complexes with initial tether lengths >500 nm moving at unloaded speeds >3 nt/s and that reached the clamp limit were selected for analysis. With the clamp active, a stall was operationally defined as beginning at the point where the velocity dropped below ∼1.5 nucleotides per second (the experimental drift limit) for more than 10 s. To minimize any sticking of DNA tethers to the coverglass surface, these experiments were conducted in flow chambers treated with blocking proteins, as described (2, 3, 9).
  • 20
    • 3643130449 scopus 로고    scopus 로고
    • note
    • We reported previously an RNAP stall force of at least 14 pN. Several lines of evidence suggested that this number may underestimate the actual value (2). First, about 20% of beads drawn by RNAP complexes were not stopped at the fixed laser powers used and escaped from the trap, permitting only a lower bound of 15 to 20 pN to be placed on their stall force. Second, long-term exposure to high levels of laser light damaged a significant proportion of complexes, leading to irreversible stalls (that is, movement that did not recover when the laser power was subsequently reduced). Third, many stalls happened abruptly, as the force increased beyond some threshold, whereas a minority of stalls took place more gradually [see figure 2C of (2)]. This suggested that the stall force might be a function of the particular nucleotide sequence being transcribed. Because force mounted slowly as RNAP traversed comparatively long segments of DNA (typically ∼500 base pairs or more), an abrupt stall might correspond to the enzyme reaching a point on the template with a lower intrinsic stall force than its previous positions. Assuming this interpretation to be correct, stall force data acquired for slowly developing loads (at low trap stiffness) would tend to produce biased estimates, preferentially sampling lower values. The feedback system developed here was designed to circumvent these difficulties (Fig. 1D). Once a moving bead reaches a displacement corresponding to some preset limit, the intensity of the laser light is actively servo-controlled (by means of an acousto-optic modulator) to prevent further motion. This clamps the bead at the limit (9), an arrangement that affords several advantages. First, photodamage is minimized; the trap light intensity is kept as low as possible until it becomes necessary to increase it, reducing the overall exposure of RNAP to the laser light by an estimated factor of five relative to earlier work. Higher peak powers are achieved without subjecting molecules to sustained exposure. As a result, all moving beads are now stalled without escaping from the trap. Second, the dynamic response of the system is dramatically improved. Force can be recorded with millisecond resolution (2, 9), and RNAP can be halted within a matter of seconds, 5-to 40-fold faster. Because the DNA tether can be readily pretensioned by the optical trap, the elastic compliance is decreased. With a taut, ∼3-kbp DNA tether, bead equilibrium position can be determined with a precision of ∼2 nm, corresponding to ∼6 base pairs, with 50 ms time resolution (or better). Third, the rapid response of the feedback system stalls the enzyme before it travels as far, leading to a less biased sampling of stall force along the template.
  • 21
    • 0003458038 scopus 로고
    • Springer-Verlag, New York
    • Records of bead position were converted to DNA contour length by a variation of the method described (9), taking advantage of the fact that many transcription complexes stopped permanently on the DNA template at the end of experimental runs. Complexes with final tether lengths >300 nm were selected for analysis. With the bead held in the trap, the microscope stage was moved in a preprogrammed fashion, and both the length and elastic properties of the residual tether were measured directly. Using both the final tether length and the force-extension relationship for the DNA molecule, in combination with time records for bead displacement and trap stiffness from the earlier experimental run, the motion of RNAP along the DNA template required to produce such records could be computed. Computed displacements were converted directly to transcript size by assuming that no slippage occurred and that one base pair corresponds to 0.338 nm [W. Saenger, Principles of Nucleic Acid Structure (Springer-Verlag, New York, 1988)].
    • (1988) Principles of Nucleic Acid Structure
    • Saenger, W.1
  • 22
    • 0019471751 scopus 로고
    • R. R. Reisbig and J. E. Hearst, Biochemistry 20, 1907 (1981); K. M. Arndt and M. J. Chamberlin, J. Mol. Biol. 213, 79 (1990); C. L. Chan and R. Landick, in Transcription: Mechanisms and Regulation, R. C. Conaway and J. W. Conaway, Eds. (Raven Press, New York, 1994), vol. 3, pp. 297-321.
    • (1981) Biochemistry , vol.20 , pp. 1907
    • Reisbig, R.R.1    Hearst, J.E.2
  • 23
    • 0025294195 scopus 로고
    • R. R. Reisbig and J. E. Hearst, Biochemistry 20, 1907 (1981); K. M. Arndt and M. J. Chamberlin, J. Mol. Biol. 213, 79 (1990); C. L. Chan and R. Landick, in Transcription: Mechanisms and Regulation, R. C. Conaway and J. W. Conaway, Eds. (Raven Press, New York, 1994), vol. 3, pp. 297-321.
    • (1990) J. Mol. Biol. , vol.213 , pp. 79
    • Arndt, K.M.1    Chamberlin, M.J.2
  • 24
    • 0019471751 scopus 로고
    • R. C. Conaway and J. W. Conaway, Eds. Raven Press, New York
    • R. R. Reisbig and J. E. Hearst, Biochemistry 20, 1907 (1981); K. M. Arndt and M. J. Chamberlin, J. Mol. Biol. 213, 79 (1990); C. L. Chan and R. Landick, in Transcription: Mechanisms and Regulation, R. C. Conaway and J. W. Conaway, Eds. (Raven Press, New York, 1994), vol. 3, pp. 297-321.
    • (1994) Transcription: Mechanisms and Regulation , vol.3 , pp. 297-321
    • Chan, C.L.1    Landick, R.2
  • 25
    • 3643074275 scopus 로고    scopus 로고
    • note
    • Pauses were removed by applying a threshold template to the velocity data, V(t), and deleting those intervals during which average speed (computed as described) fell below 50% of the template value at any given time. The remaining portions of the record were then concatenated to form a new record. The threshold template function was fixed in advance for each run and consisted of two sequential linear segments: one constant, the next tapering linearly from this constant value to zero at some fixed point in the record. The amplitude of the constant, the slope of the linear taper, and the fixed intercept point were chosen empirically. Taken together, the two line segments roughly approximate the shape of V(t) curves. In principle, it is impossible to distinguish between transient transcriptional pausing and mechanical stalling in the limit that complexes are halted by an external load, based solely on velocity data. Furthermore, uncertainties due to noise and drift make it difficult to ascertain exactly when stall is achieved (11). For these reasons, the shapes of F-V curves are poorly determined near their x-intercepts (zero velocity).
  • 26
    • 3643064809 scopus 로고    scopus 로고
    • note
    • The calculated efficiencies neglect energetic contributions from RNA folding.
  • 29
    • 3643067963 scopus 로고    scopus 로고
    • note
    • The large heterogeneity in stall force may reflect several contributions. (i) Complexes were oriented randomly on the surface with respect to the direction of the applied load, and the dependence upon angle may be significant; (ii) Stall forces may depend on the template sequence; (iii) Heterogeneity may exist in the properties of nominally identical RNAP molecules.
  • 30
    • 3643091903 scopus 로고    scopus 로고
    • note
    • In principle, elongation by RNAP can be arrested by any of a variety of physical mechanisms. (i) The applied load could reversibly halt progress, via mechanochemical coupling in the enzyme; (ii) the load could place the enzyme in a transcriptional pause state; (iii) the load could temporarily inhibit the enzyme, placing it in a state that is biochemically distinct from a pause; or (iv) the enzyme could become irreversibly damaged, either directly by the applied load or by exposure to the laser light. In these and earlier experiments (2), both reversible and irreversible stalls were found, and reversible stalls required the same average force within error as irreversible stalls, suggesting that most damage occurred subsequent to stall. Distinguishing among mechanisms (i) through (iii) is nontrivial and will require additional biochemical, as well as mechanical, data.
  • 31
    • 0027761137 scopus 로고
    • D. A. Erie, O. Hajiseyedjavadi, M. C. Young, P. H. von Hippel, Science 262, 867 (1993); H. Matsuzaki, G. A. Kassavetis, E. P. Geiduschek, J. Mol. Biol. 235, 1173 (1994); C. Chan, D. Wang, R. Landick, ibid. 268, 54 (1997).
    • (1993) Science , vol.262 , pp. 867
    • Erie, D.A.1    Hajiseyedjavadi, O.2    Young, M.C.3    Von Hippel, P.H.4
  • 32
    • 0028224347 scopus 로고
    • D. A. Erie, O. Hajiseyedjavadi, M. C. Young, P. H. von Hippel, Science 262, 867 (1993); H. Matsuzaki, G. A. Kassavetis, E. P. Geiduschek, J. Mol. Biol. 235, 1173 (1994); C. Chan, D. Wang, R. Landick, ibid. 268, 54 (1997).
    • (1994) J. Mol. Biol. , vol.235 , pp. 1173
    • Matsuzaki, H.1    Kassavetis, G.A.2    Geiduschek, E.P.3
  • 33
    • 0031585994 scopus 로고    scopus 로고
    • D. A. Erie, O. Hajiseyedjavadi, M. C. Young, P. H. von Hippel, Science 262, 867 (1993); H. Matsuzaki, G. A. Kassavetis, E. P. Geiduschek, J. Mol. Biol. 235, 1173 (1994); C. Chan, D. Wang, R. Landick, ibid. 268, 54 (1997).
    • (1997) J. Mol. Biol. , vol.268 , pp. 54
    • Chan, C.1    Wang, D.2    Landick, R.3
  • 34
    • 0030950638 scopus 로고    scopus 로고
    • N. Komissarova and M. Kashlev, J. Biol. Chem. 272, 15329 (1997); E. Nudler, A. Mustaev, E. Lukhtanov, A. Goldfarb, Cell 89, 33 (1997).
    • (1997) J. Biol. Chem. , vol.272 , pp. 15329
    • Komissarova, N.1    Kashlev, M.2
  • 37
    • 3643058530 scopus 로고    scopus 로고
    • note
    • More complex models for movement can be entertained, including those where the tight-coupling assumption is violated. Such loosely coupled enzymatic schemes are not parsimonious, however, as they involve greater numbers of free parameters.
  • 42
    • 3643101270 scopus 로고    scopus 로고
    • note
    • eff/F. Normalization through Eq. 10 permits F-V curves with different apparent δ to be compared, yielding an average value.
  • 43
    • 3643079426 scopus 로고    scopus 로고
    • note
    • Supported by grants from the National Institute of General Medical Sciences (R.L., J.G., and S.M.B), and from NSF and the W. M. Keck Foundation (S.M.B). M.D.W. was supported by a Damon Runyon-Walter Winchell Cancer Research fund postdoctoral fellowship. M.J.S. was supported by an American Heart Association predoctoral fellowship and a Proctor Honorific Fellowship from Princeton University. S.M.B. and M.D.W. thank S. Gross, L. Satterwhite, and K. Visscher for helpful discussions and advice, and K. Neuman for comments on an early draft of the manuscript. A movie of the tether-particle assay can be viewed at www.rose.brandeis.edu/users/gelles/ stall/


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.