One-dimensional Diffusion of Proteins along DNA

Evidence for sliding of proteins along DNA has been provided by many kinetic studies, but single-molecule-based measurements have uncovered distinct problems, the solutions of which may lead us to an understanding of new mechanisms for gene regulation. Furthermore, they reveal a deep problem lying between chemistry and physics regarding the seemingly simple binding between DNA and protein. Single-molecule dynamics provides a tool to solve this problem without prejudgments or unsound assumptions. Single-molecule dynamics is the most powerful, and probably the only direct, method for analyzing a biological phenomenon in which the history of a single molecule appears at macroscopic level. The long-standing question as to whether or not proteins slide along nonspecific parts of DNA provides an excellent example of the power of the new method and its complementarity to classic kinetics. The sliding of a protein (a microscopic history) can macroscopically enhance the formation of specific complexes, as described below (see also Ref. 1von Hippel P.H. Berg O.G. J. Biol. Chem. 1989; 264: 675-678Abstract Full Text PDF PubMed Google Scholarfor review). The histories of individual molecules have traditionally been considered unimportant in chemistry, because the differences in histories of different molecules are normally obliterated due to numerous random collisions with solvent molecules. The resulting group of molecules with a unified average history is considered as a single chemical species. In practice, however, a chemical species is usually arbitrarily defined, and such a theoretical uncertainty causes a serious problem in kinetics, because kinetic analysis is based on hypotheses of mechanisms. Direct observation of individual molecules, on the other hand, requires no theoretical assumptions. This is the most important reason for the application of single-molecule dynamics to a problem like sliding. The limitation of space here has urged exclusion of the sliding of DNA clumps and of the energy-driven translocation of enzymes such as helicases and RNA polymerase being engaged in RNA synthesis; the successful single-molecule studies of the latter have been recently reviewed (2Gelles J. Landick R. Cell. 1998; 93: 13-16Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). To avoid serious confusion due to the variable terminology used in past discussions of protein sliding, specific terms should first be defined clearly. One-dimensional diffusion of a protein here includes all mechanisms of translocation along a single DNA molecule that do not involve a free state of the protein. These mechanisms are classified into sliding and intersegment transfer (Fig.1). The latter is here supposed to require at least two DNA binding sites on the protein molecule as in the general theory (3Berg O.G. Winter R.B. von Hippel P.H. Biochemistry. 1981; 20: 6929-6948Crossref PubMed Scopus (967) Google Scholar, 4Berg O.G. Ehrenberg M. Biophys. Chem. 1982; 15: 41-51Crossref PubMed Scopus (47) Google Scholar), excluding a transient interaction as a secondary binding. Sliding and hopping are taken to imply, respectively, a helical movement due to tracking a groove of DNA or a non-helical movement parallel to the DNA axis (5Jeltsch A. Alves J. Wolfs H. Maass G. Pingoud A. Biochemistry. 1994; 33: 10215-10219Crossref PubMed Scopus (83) Google Scholar, 6Berkhout B. van Wamel J. J. Biol. Chem. 1996; 271: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), although they have been differently defined theoretically (3Berg O.G. Winter R.B. von Hippel P.H. Biochemistry. 1981; 20: 6929-6948Crossref PubMed Scopus (967) Google Scholar). These distinctions are sacrificed here for simplicity, because a simple microscopic observation cannot distinguish them. To visualize movement of a single molecule of protein using a commercially available fluorescent microscope, the molecule must be made strongly fluorescent. This can be done by attaching several tens of fluorophores per molecule (7Shimamoto N. Kabata H. Kurosawa O. Washizu M. Lilley D. Heumann H. Suck D. Structural Tools for the Analysis of Protein-Nucleic Acid Complexes. Birkhäuser Verlag AG, Basel1992: 241-253Google Scholar, 8Kabata H. Kurosawa O. Arai I. Washizu M. Margarson S.A. Glass R.E. Shimamoto N. Science. 1993; 262: 1561-1563Crossref PubMed Scopus (240) Google Scholar). The clearest visual assay of sliding movement is to let DNA take up a special geometry as in Fig. 2 A and to detect the traces of protein molecules moving with the same geometry. A DNA concentration of 10–100 μg/ml is required to observe binding events at a significant frequency. These requirements were satisfied by applying dielectrophoresis to align extended DNA molecules in parallel (9Washizu M. Kurosawa O. Arai I. Suzuki S. Shimamoto N. IEEE Trans. Ind. Appl. 1995; 31: 447-456Crossref Scopus (156) Google Scholar). Fluorescently labeled Escherichia coli RNA polymerase holoenzyme was injected so as to flow at an angle across the array of DNA molecules (8Kabata H. Kurosawa O. Arai I. Washizu M. Margarson S.A. Glass R.E. Shimamoto N. Science. 1993; 262: 1561-1563Crossref PubMed Scopus (240) Google Scholar). Linear motions parallel to DNA were observed in half of the traces passing through the DNA region (Fig. 2 A). The linear motions disappeared when holoenzyme was preincubated with heparin or with a short DNA fragment harboring a strong promoter. Furthermore, neither the IgG used for fluorescent labeling nor microcrystals of rhodamine showed linear motions. These negative controls, together with the quantitative agreement between the lifetimes of the observed sliding complexes and those previously measured for nonspecific holoenzyme-DNA complexes (10Park C.S. Hillel Z. Wu C.-W. J. Biol. Chem. 1982; 257: 6944-6949Abstract Full Text PDF PubMed Google Scholar), prove that the observed movement is a true sliding of holoenzyme along DNA and not a hydrodynamic artifact such as rectification of flow in the DNA region. The sliding complex is the only complex observed in nonspecific regions of the DNA, and stably bound complexes form only at the promoter. Moreover, a typical bacterial repressor, Pseudomonas putidaCamR, which is a small homodimeric protein with a helix-turn-helix DNA binding motif, shows very similar movements. 1H. Kabata and N. Shimamoto, unpublished results. These results suggest that sliding is a general property of DNA-binding proteins, although not all might be able to slide. The flow introduced in the above assay, or asymmetric collisions with solvent molecules, converts otherwise bidirectional sliding movements into a large unidirectional travel of several micrometers. The length of travel should not be confused with the sliding distance, which is defined as the mean size of DNA segment scanned per binding event (in the absence of flow), and has been kinetically estimated as 350–1000 base pairs for RNA polymerase (11Singer P. Wu C.-W. J. Biol. Chem. 1987; 262: 14178-14189Abstract Full Text PDF PubMed Google Scholar, 12Ricchetti M. Metzger W. Heumann H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4610-4614Crossref PubMed Scopus (47) Google Scholar). Atomic force microscopy can measure the movement of single molecules trapped on a flat surface at a resolution smaller than 1 nm. By fixing DNA on a mica surface, the random displacements due to sliding ofAspergillus nidulans photolyase have been directly detected (13van Noort S.J. van der Werf K.O. Eker A.P. Wyman C. de Grooth B.G. van Hulst N.F. Greve J. Biophys J. 1998; 74: 2840-2849Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The drawback of this method is slowing down of the movements of molecules because of interaction with a surface. The relationship between the new single-molecule-based techniques and classic kinetics resembles that between the new techniques developed in molecular biology and classical genetics, which have become fused and essential to modern biology. Kinetics can compensate for the defects of single-molecule dynamics, namely the heavy modification used for visualizing and detecting target molecules, and the unusual experimental conditions. One-dimensional diffusion along DNA was first deduced from the finding that E. coli LacI binds to its operators on a 48-kilobase pair DNA 100-fold faster than by the fastest three-dimensional diffusion theoretically predicted from the sizes of the reactants (14Riggs A.D. Bougeois S. Cohn M. J. Mol. Biol. 1970; 53: 401-417Crossref PubMed Scopus (640) Google Scholar). A similar controversy was found in the binding of E. coliRNA polymerase (15Belinstev B.N. Zavriev S.K. Shemyakin M.F. Nucleic Acids Res. 1980; 8: 1391-1404Crossref PubMed Scopus (22) Google Scholar). Since then kinetic evidence for sliding has been accumulated by using the assays shown in Fig. 2, B–F. The black box nature of kinetics would blur the meanings of evaluated kinetic parameters, but the most important qualitative conclusion, the existence of sliding, is very likely to be correct for the following proteins: restriction endonuclease EcoRI (5Jeltsch A. Alves J. Wolfs H. Maass G. Pingoud A. Biochemistry. 1994; 33: 10215-10219Crossref PubMed Scopus (83) Google Scholar, 16Ehbrecht H.-J. Pingoud A. Urebenbake G. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar, 17Terry B.J. Jack W.E. Modrich P. J. Biol. 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Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar,22Nardone G. George J. Chirikjian J.G. J. Biol. Chem. 1986; 261: 12128-12133Abstract Full Text PDF PubMed Google Scholar), EcoRV (21Jeltsch A. Wenz C. Stahl F. Pingoud A. EMBO J. 1996; 15: 5104-5111Crossref PubMed Scopus (84) Google Scholar), E. coli RNA polymerase (7Shimamoto N. Kabata H. Kurosawa O. Washizu M. Lilley D. Heumann H. Suck D. Structural Tools for the Analysis of Protein-Nucleic Acid Complexes. Birkhäuser Verlag AG, Basel1992: 241-253Google Scholar, 8Kabata H. Kurosawa O. Arai I. Washizu M. Margarson S.A. Glass R.E. Shimamoto N. Science. 1993; 262: 1561-1563Crossref PubMed Scopus (240) Google Scholar,10Park C.S. Hillel Z. Wu C.-W. J. Biol. Chem. 1982; 257: 6944-6949Abstract Full Text PDF PubMed Google Scholar, 11Singer P. Wu C.-W. J. Biol. Chem. 1987; 262: 14178-14189Abstract Full Text PDF PubMed Google Scholar, 12Ricchetti M. Metzger W. Heumann H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4610-4614Crossref PubMed Scopus (47) Google Scholar, 23Singer P. Wu C.-W. J. Biol. Chem. 1988; 263: 4208-4214Abstract Full Text PDF PubMed Google Scholar), LacI (24Ruusala T. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4903-4907Crossref PubMed Scopus (64) Google Scholar, 25Barkley M.D. Biochemistry. 1981; 20: 3833-3842Crossref PubMed Scopus (89) Google Scholar, 26Winter R.B. Berg O.G. von Hippel P.H. Biochemistry. 1981; 20: 6961-6967Crossref PubMed Scopus (460) Google Scholar, 27Berg O.G. Blomberg C. Biophys. Chem. 1978; 8: 271-280Crossref PubMed Scopus (52) Google Scholar, 28Lohman T.M. deHaseth P.L. Record M.T. Biophys. Chem. 1978; 8: 281-294Crossref PubMed Scopus (75) Google Scholar, 29Khoury A.M. Lee H.J. Lillis M. Lu P. Biochim. Biophys. Acta. 1990; 1087: 55-60Crossref PubMed Scopus (27) Google Scholar, 30Hsieh M. Brenowitz M. J. Biol. Chem. 1997; 272: 22092-22096Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), GalR (30Hsieh M. Brenowitz M. J. Biol. Chem. 1997; 272: 22092-22096Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), EcoRI methylase (31Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2209-2217Crossref PubMed Scopus (47) Google Scholar,32Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2201-2208Crossref PubMed Scopus (57) Google Scholar), BamHI methylase (22Nardone G. George J. Chirikjian J.G. J. Biol. Chem. 1986; 261: 12128-12133Abstract Full Text PDF PubMed Google Scholar), UvrABC (33Gruskin E.A. Lloyd R.S. J. Biol. Chem. 1988; 263: 12738-12743Abstract Full Text PDF PubMed Google Scholar), λ Cro repressor (34Kim J.G. Takeda Y. Matthews B.W. Anderson W.F. J. Mol. Biol. 1987; 196: 149-158Crossref PubMed Scopus (116) Google Scholar), and T4 endonuclease V (35Lloyd R.S. Hanawalt P.C. Dodson M.L. Nucleic Acids Res. 1980; 8: 5113-5127Crossref PubMed Scopus (72) Google Scholar, 36Gruskin E.A. Lloyd R.S. J. Biol. Chem. 1988; 263: 12728-12737Abstract Full Text PDF PubMed Google Scholar). The kinetic length effects (Fig.2 B) observed for LacI were once attributed to intersegment transfer (37Fickert R. Mueller-Hill B. J. Mol. Biol. 1992; 226: 59-68Crossref PubMed Scopus (65) Google Scholar), but more critically designed experiments (Fig.2 D) established the coexistence of sliding (24Ruusala T. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4903-4907Crossref PubMed Scopus (64) Google Scholar). Several quantitative techniques to detect DNA-protein complexes have been used in the kinetic test for sliding. The filter-binding technique has been widely used (14Riggs A.D. Bougeois S. Cohn M. J. Mol. Biol. 1970; 53: 401-417Crossref PubMed Scopus (640) Google Scholar, 15Belinstev B.N. Zavriev S.K. Shemyakin M.F. Nucleic Acids Res. 1980; 8: 1391-1404Crossref PubMed Scopus (22) Google Scholar, 18Jack W.E. Terry B.J. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4010-4014Crossref PubMed Scopus (144) Google Scholar, 24Ruusala T. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4903-4907Crossref PubMed Scopus (64) Google Scholar, 26Winter R.B. Berg O.G. von Hippel P.H. Biochemistry. 1981; 20: 6961-6967Crossref PubMed Scopus (460) Google Scholar, 29Khoury A.M. Lee H.J. Lillis M. Lu P. Biochim. Biophys. Acta. 1990; 1087: 55-60Crossref PubMed Scopus (27) Google Scholar, 31Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2209-2217Crossref PubMed Scopus (47) Google Scholar, 34Kim J.G. Takeda Y. Matthews B.W. Anderson W.F. J. Mol. Biol. 1987; 196: 149-158Crossref PubMed Scopus (116) Google Scholar, 37Fickert R. Mueller-Hill B. J. Mol. Biol. 1992; 226: 59-68Crossref PubMed Scopus (65) Google Scholar) but is open to misinterpretation if there are two or more types of complexes that are trapped with different efficiencies (37Fickert R. Mueller-Hill B. J. Mol. Biol. 1992; 226: 59-68Crossref PubMed Scopus (65) Google Scholar). If practicable, the gel shift assay (38Fried M.G. Crothers D.M. Nucleic Acids Res. 1981; 9: 6505-6525Crossref PubMed Scopus (1686) Google Scholar, 39Garner M.M. Revzin A. Nucleic Acids Res. 1981; 9: 3047-3060Crossref PubMed Scopus (1209) Google Scholar) is an excellent choice (24Ruusala T. Crothers D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4903-4907Crossref PubMed Scopus (64) Google Scholar,37Fickert R. Mueller-Hill B. J. Mol. Biol. 1992; 226: 59-68Crossref PubMed Scopus (65) Google Scholar, 40Winter R.B. von Hippel P.H. Biochemistry. 1981; 20: 6948-6960Crossref PubMed Scopus (233) Google Scholar), because it can be made highly quantitative by introducing a competition between two DNA fragments (41Yang S. Nash H.A. EMBO J. 1995; 14: 6292-6300Crossref PubMed Scopus (86) Google Scholar). An up-to-date method, surface plasmon resonance, needs special care when applied to rapid reactions such as the binding of protein and DNA. The binding kinetics can be disturbed by mass transportation to the surface of detection and by rebinding events (42Schuck P. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 539-564Crossref Scopus (545) Google Scholar). Rapid mixing techniques have been successfully combined with DNase I footprinting (30Hsieh M. Brenowitz M. J. Biol. Chem. 1997; 272: 22092-22096Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and UV flash photo-cross-linking (Fig. 2 E) (10Park C.S. Hillel Z. Wu C.-W. J. Biol. Chem. 1982; 257: 6944-6949Abstract Full Text PDF PubMed Google Scholar, 11Singer P. Wu C.-W. J. Biol. Chem. 1987; 262: 14178-14189Abstract Full Text PDF PubMed Google Scholar, 23Singer P. Wu C.-W. J. Biol. Chem. 1988; 263: 4208-4214Abstract Full Text PDF PubMed Google Scholar). Notably the latter can directly detect nonspecific complexes. The formation of a specific complex of an enzyme can be kinetically monitored by its catalytic consequences, provided that the catalytic reaction is more rapid than the breakdown of the specific complex, a condition called diffusion control (16Ehbrecht H.-J. Pingoud A. Urebenbake G. Maass G. Gualerzi C. J. Biol. Chem. 1985; 260: 6160-6166Abstract Full Text PDF PubMed Google Scholar, 18Jack W.E. Terry B.J. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4010-4014Crossref PubMed Scopus (144) Google Scholar, 20Jeltsch A. Fritz A. Alves J. Wolfs H. Pingoud A. Anal. Biochem. 1993; 213: 234-240Crossref PubMed Scopus (96) Google Scholar, 22Nardone G. George J. Chirikjian J.G. J. Biol. Chem. 1986; 261: 12128-12133Abstract Full Text PDF PubMed Google Scholar, 32Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2201-2208Crossref PubMed Scopus (57) Google Scholar). A better designed enzyme assay shown in Fig. 2 F is free from this limitation. Interestingly a groove-tracking type of sliding was shown for EcoRI (5Jeltsch A. Alves J. Wolfs H. Maass G. Pingoud A. Biochemistry. 1994; 33: 10215-10219Crossref PubMed Scopus (83) Google Scholar) and BssHII (6Berkhout B. van Wamel J. J. Biol. Chem. 1996; 271: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) endonucleases by this assay. Notably this processivity assay is also possible in vivo (21Jeltsch A. Wenz C. Stahl F. Pingoud A. EMBO J. 1996; 15: 5104-5111Crossref PubMed Scopus (84) Google Scholar, 33Gruskin E.A. Lloyd R.S. J. Biol. Chem. 1988; 263: 12738-12743Abstract Full Text PDF PubMed Google Scholar, 36Gruskin E.A. Lloyd R.S. J. Biol. Chem. 1988; 263: 12728-12737Abstract Full Text PDF PubMed Google Scholar). Sliding along DNA obviously can kinetically affect a biological process through the acceleration of association, if bimolecular association is the rate-limiting step of the process. This situation, however, may only apply to limited cases in vivo. For example, transcription initiation includes several time-consuming elementary reaction steps. As a result the association of RNA polymerase with a promoter is rarely rate-limiting, making the acceleration of association by sliding less significant. However, sliding may be more general in the production of processivity, as evidenced in restriction (21Jeltsch A. Wenz C. Stahl F. Pingoud A. EMBO J. 1996; 15: 5104-5111Crossref PubMed Scopus (84) Google Scholar, 31Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2209-2217Crossref PubMed Scopus (47) Google Scholar, 32Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2201-2208Crossref PubMed Scopus (57) Google Scholar) and repair (33Gruskin E.A. Lloyd R.S. J. Biol. Chem. 1988; 263: 12738-12743Abstract Full Text PDF PubMed Google Scholar, 36Gruskin E.A. Lloyd R.S. J. Biol. Chem. 1988; 263: 12728-12737Abstract Full Text PDF PubMed Google Scholar) of DNA. If a protein slides from its specific site into nonspecific sites (Fig. 3 A), longer DNA will accelerate the overall dissociation from the specific site. This length effect compensates the kinetic length effect on association (Fig.2 B), and binding affinity will be unchanged. Indeed such absence of the enhancement of affinity by sliding has been reported forEcoRI (18Jack W.E. Terry B.J. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4010-4014Crossref PubMed Scopus (144) Google Scholar) and Cro repressor (34Kim J.G. Takeda Y. Matthews B.W. Anderson W.F. J. Mol. Biol. 1987; 196: 149-158Crossref PubMed Scopus (116) Google Scholar). However, there are several cases showing enhancement of affinity by the extension of DNA (Fig. 3 C); the observed enhancements are 20-fold for EcoRI methylase (31Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2209-2217Crossref PubMed Scopus (47) Google Scholar, 32Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2201-2208Crossref PubMed Scopus (57) Google Scholar), 10-fold for human immunodeficiency virus type 1 integrase (43Lee S.P. Censullo M.L. Kim H.G. Han M.K. Biochemistry. 1995; 34: 10215-10223Crossref PubMed Scopus (22) Google Scholar), more than 100-fold for CamR,1 and 10–30-fold for LacI (26Winter R.B. Berg O.G. von Hippel P.H. Biochemistry. 1981; 20: 6961-6967Crossref PubMed Scopus (460) Google Scholar, 29Khoury A.M. Lee H.J. Lillis M. Lu P. Biochim. Biophys. Acta. 1990; 1087: 55-60Crossref PubMed Scopus (27) Google Scholar). Interestingly the existence of sliding by RNA polymerase was detected by preferential occupancies among several identical promoters on the same DNA, using a design similar to that shown in Fig. 2 M. Metzger W. Heumann H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4610-4614Crossref PubMed Scopus (47) Google Scholar). DNA up to base pairs in length affinity for its promoter in an although this applied only when it was of the promoter. These that an extended region of nonspecific DNA can as an to protein for binding to a specific site. To this effect without making theoretical the movement traces of molecules of and RNA polymerase were following dissociation from specific sites in the of of the traces of showed dissociation into or sliding movements small to be by the microscope, to dissociation In the traces of RNA polymerase showed sliding the dissociation (Fig. 3 (8Kabata H. Kurosawa O. Arai I. Washizu M. Margarson S.A. Glass R.E. Shimamoto N. Science. 1993; 262: 1561-1563Crossref PubMed Scopus (240) Google Scholar) as as the dissociation of will be less than association by in DNA with the observed large enhancement of its The of the small effect found for RNA polymerase M. Metzger W. Heumann H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4610-4614Crossref PubMed Scopus (47) Google Scholar) can also be along If RNA polymerase slides the promoter more in an than a because of the of the the extension of DNA would enhance the promoter affinity more than extension making the effect An for the effect is that the length of DNA the specific complex, a transient secondary which the specific If longer DNA the complex 100-fold by this dissociation only from a of complexes that are not the overall dissociation to of that in the absence of if is rate-limiting in the overall dissociation This however, the observation that the dissociation is of DNA length in the of EcoRI methylase (31Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2209-2217Crossref PubMed Scopus (47) Google Scholar). Furthermore, this cannot the asymmetric effect with RNA polymerase or the absence of sliding dissociation of sliding is the as the of the If the of sliding would be more important than previously for specific sites be by limitation of sliding movements In EcoRI endonuclease is by protein complexes the as as unusual DNA such as sites and (5Jeltsch A. Alves J. Wolfs H. Maass G. Pingoud A. Biochemistry. 1994; 33: 10215-10219Crossref PubMed Scopus (83) Google Scholar). This is at a in the true which be in many gene mechanisms such as in In most kinetic studies nonspecific complexes are supposed to form a single chemical a free nonspecific complex specific This has been made the of a chemical which that of the species has an of nonspecific complexes at and sites have different of sliding into a specific the above in effect the of sliding from the a nonspecific complex at on DNA be considered a distinct chemical as has been supposed in a general theory (see the of Ref. O.G. Winter R.B. von Hippel P.H. Biochemistry. 1981; 20: 6929-6948Crossref PubMed Scopus (967) Google Scholar) O.G. Ehrenberg M. Biophys. Chem. 1982; 15: 41-51Crossref PubMed Scopus (47) Google Scholar). If the between nonspecific should be to collisions with solvent molecules to the histories of individual protein molecules they at a nonspecific site. the other the single-molecule assay that this is so small that the of sliding is half of the parallel of flow (8Kabata H. Kurosawa O. Arai I. Washizu M. Margarson S.A. Glass R.E. Shimamoto N. Science. 1993; 262: 1561-1563Crossref PubMed Scopus (240) Google Scholar). In other polymerase molecules along DNA of nonspecific This may or may not the distinct species at but the that a group of nonspecific complexes the sliding would more a chemical species than at the true chemical of nonspecific complexes is not The problem of the as has been (31Surby M.A. Reich N.O. Biochemistry. 1996; 35: 2209-2217Crossref PubMed Scopus (47) Google Scholar), is a to a of The of which is from the of and the of chemical an and between a specific complex and the free in if two two chemical species that are directly by an elementary chemical The of a specific complex by the effect should the from the specific In the from the free should be because and are of the the is in the chemical of the This be in two the existence of an chemical species. The complex is to be but cannot experimental as In an the chemical species of the specific complex is defined to the above so that it includes all complexes the sliding from the specific site. In an space a DNA which can be the is to and the protein molecules from the extended specific site this space and in As a distinct chemical the protein in this DNA can with free protein and with the extended specific the length of DNA up to the sliding would also the DNA and the from free state into the The the because in it to the into and the from the specific which are by the a space of this was found in rapid UV photo-cross-linking studies of sliding (10Park C.S. Hillel Z. Wu C.-W. J. Biol. Chem. 1982; 257: 6944-6949Abstract Full Text PDF PubMed Google Scholar, 11Singer P. Wu C.-W. J. Biol. Chem. 1987; 262: 14178-14189Abstract Full Text PDF PubMed Google Scholar, 23Singer P. Wu C.-W. J. Biol. Chem. 1988; 263: 4208-4214Abstract Full Text PDF PubMed Google Scholar). RNA polymerase in a rapid flow was to this space in a state such that was and the of such a space was for experiments would prove or this in the and they are very likely to be single-molecule because they should be designed so as to be free from chemical assumptions. The above that the of molecules may not with macroscopic not only in but also in the chemistry and biology of seemingly simple DNA-protein I R. S. of for of the and J. C. P. Modrich and M. of for