FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2056588849> ?p ?o ?g. }

• W2056588849 endingPage "30458" @default.
• W2056588849 startingPage "30451" @default.
• W2056588849 abstract "The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (−4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, E+D⇄K1EDa⇄k−2k2EDb where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Φ10 and Φ3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k−2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k−2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Φ3.8 a weaker promoter relative to Φ10. The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (−4 to +4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, E+D⇄K1EDa⇄k−2k2EDb where the RNA polymerase forms an initial weak EDa complex rapidly with an equilibrium association constant K1. The EDa complex then undergoes a conformational change to EDb, wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of EDa to EDb is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Φ10 and Φ3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, kon (K1k2), and the dissociation rate constant, koff (k−2), and thermodynamics, the equilibrium constants of the two steps (K1 and k2/k−2) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower kon, the 4-fold higher koff, and the 2-5-fold weaker equilibrium interactions together make Φ3.8 a weaker promoter relative to Φ10. Bacteriophage T7 RNA polymerase is a 98-kDa single subunit polymerase that catalyzes synthesis of RNA complementary in sequence to the template DNA (1Chamberlin, M. J., Ryan, T., (1983) The Enzymes, (Boyer, P. D., ed), Vol. 15, pp. 87–108, Academic Press, New York.Google Scholar). The phage enzymes are among the simplest RNA polymerases known, as no accessory proteins are necessary for specific initiation, elongation, or termination of transcription (2Davanloo P. Rosenberg A.H. Dunn J.J. Studier F.W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2035-2039Crossref PubMed Scopus (731) Google Scholar, 3Kotani H. Ishizaki Y. Hiraoka N. Obayashi A. Nucleic Acids Res. 1987; 15: 2653-2664Crossref PubMed Scopus (57) Google Scholar). The 17 promoters of bacteriophage T7 direct specific initiation of RNA synthesis that occurs in a rapid and processive manner (4Martin C.T. Coleman J.E. Biochemistry. 1987; 26: 2690-2696Crossref PubMed Scopus (118) Google Scholar, 5Ikeda R.A. Lin A.C. Clarke J. J. Biol. Chem. 1992; 267: 2640-2649Abstract Full Text PDF PubMed Google Scholar). Due to their simplicity these enzymes serve as model systems to understand, in depth, the mechanisms of transcription initiation, elongation, or termination. Initiation of transcription occurs by recognition and binding of the RNA polymerase to a promoter DNA sequence. This event is recognized as one of the important steps at which transcription and gene expression is regulated. The 17 bacteriophage T7 promoters share consensus sequence from −17 to +6 position relative to the transcription start site at +1 (6Dunn J.J. Studier F.W. J. Mol. Biol. 1983; 166: 477-535Crossref PubMed Scopus (985) Google Scholar). The class III gene promoters of T7 are absolutely conserved in DNA sequence, whereas the class II gene promoters differ at a number of positions within the consensus sequence. The sequence of the promoter DNA is a primary factor that determines the strength of the promoter and the efficiency of initiation. However, the relationship between promoter DNA sequence and transcriptional efficiency is not well understood at the mechanistic level. In general, the detailed kinetics and thermodynamics of transcription are less well understood, in part, due to its complexity. The T7 promoters and the phage RNA polymerase, owing to their simplicity, should serve as a good starting model to understand the mechanism and regulation of transcription in greater detail. The present study consists of the kinetic and thermodynamic investigations of the steps involved in promoter recognition and DNA binding. The interaction of the RNA polymerase with its promoter DNAs has not been examined previously using fast kinetic methods. Since DNA binding is a fast step, it is necessary to use rapid kinetic methods to directly observe intermediate binary species that accumulate transiently during initiation. The stopped-flow methods used in this study allow us to elucidate the mechanism of DNA binding and to determine the rate and equilibrium constants of steps leading to intermediate species. To examine DNA binding in real time we use the change in the intrinsic fluorescence of protein upon promoter binding as a signal. In addition, we have taken advantage of the fluorescent properties of 2-aminopurine (2-AP) 1The abbreviations used are: 2-AP2-aminopurinedsdouble-stranded. base, an analog of dA, which can be incorporated into the promoter DNAs and used as a probe to monitor promoter opening in real time. The present studies have been carried out in the absence of the NTP substrate. Comparative studies with two natural T7 promoters, Φ10 and Φ3.8, representing strong class III and weak class II promoters, respectively, provide insights into the mechanism by which T7 RNA polymerase regulates its transcriptional efficiency at the level of DNA binding. 2-aminopurine double-stranded. T7 RNA polymerase was purified from the Escherichia coli BL21/pAR1219 cell line (kindly provided by Alan Rosenberg and Bill Studier, Brookhaven National Laboratories) (2Davanloo P. Rosenberg A.H. Dunn J.J. Studier F.W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2035-2039Crossref PubMed Scopus (731) Google Scholar). The enzyme was >95% pure after three chromatography columns consisting of SP-Sephadex, CM-Sephadex, and DEAE-Sephacel purchased from Sigma (7Grodberg J. Dunn J.J. J. Bacteriol. 1988; 170: 1245-1253Crossref PubMed Scopus (576) Google Scholar). The polymerase was stored in 50% glycerol, and buffer containing 20 mM sodium phosphate (pH 7.7), 1 mM trisodium EDTA, 1 mM dithiothreitol, 100 mM NaCl at −80°C. The concentration of the polymerase was determined by absorbance measurement at 280 nm and from its molar extinction coefficient of 1.4 × 105 M−1 cm−1 (8King G.C. Martin C.T. Pham T.T. Coleman J.E. Biochemistry. 1986; 25: 36-40Crossref PubMed Scopus (90) Google Scholar). The DNA promoters were synthesized on a Millipore Nucleic Acid synthesis system 899. DMT-deoxynucleoside (benzoyl or isobutyryl) β-cyanoethylphosphoramidites were purchased from PerSeptive Biosystems. 2-Aminopurine CE phosphoramidite and Ac-dC-CE phosphoramidite were purchased from Glen Research Corp. Ac-dC-CE phosphoramidite was used for dC incorporation in 2-AP DNA. Coupling time of 15 min for 2-AP base incorporation and ultrafast cleavage and deprotection systems were used for the synthesis of 2-AP containing DNAs (deprotection was performed with a mixture of equal volumes of ammonium hydroxide and 40% methylamine aqueous solution, following the procedure provided by Glen Research). 2-AP base was incorporated at position −3, −1, and +4 for Φ10 and Φ3.8 nontemplate strands, position −4 and −2 for Φ10 template strand, and position −4 for the Φ3.8 template strand (Fig. 1). All synthetic promoters used in this study were purified on 16% polyacrylamide, 5 M urea gels. The DNA was visualized by UV shadowing, and electroeluted from the gel using an Elutrap apparatus (Schleicher & Schuell). The concentration of purified DNA was determined by absorbance measurement at 260 nm using the following extinction coefficients (M−1 cm−1) for the bases: dA, 15,200; dC, 7050; dG, 12,010; dT, 8400. The extinction coefficient of 2-AP, at 260 nm, equal to 1000 M−1 cm−1 was used in the calculation of 2-AP DNA concentration (9Fox J.J. Wempen I. Hampton A. Doerr I.L. J. Am. Chem. Soc. 1958; 80: 1669-1675Crossref Scopus (205) Google Scholar). The double-stranded (ds) DNAs were prepared by annealing the individual single-stranded DNA strands. The exact ratio of the two single-stranded DNA strands to prepare the dsDNAs was determined routinely from titration experiments performed on an 18% native polyacrylamide gel that resolves dsDNA from the single-stranded DNAs. The fluorimetric titration experiments were performed on a Perkin Elmer LS50B Luminescence Spectrometer. The emission spectra of 2-AP containing DNAs were obtained by excitation at 315 nm (excitation slit width = 5 nm, and emission slit width = 5 nm). Fluorimetric titration experiments were performed in the reaction buffer (50 mM Tris acetate, pH 7.5, 50 mM sodium acetate, 10 mM magnesium acetate, 5 mM dithiothreitol, 0.05% Tween 20) at 25°C. The fluorescence changes recorded in all these experiments are an average of 5 measurements which were taken after at least 1 min of incubation when the readings no longer changed. A constant amount of 2-AP dsDNA (0.05 μM) was titrated against increasing concentration of T7 RNA polymerase. A control experiment was carried out in the presence of normal DNA (0.05 μM) and increasing amounts of the polymerase, and the spectra were subtracted from the above to obtain fluorescence changes due to polymerase·2-AP DNA complex formation. The stopped-flow instrument from KinTek Corp. (State College, PA) was used to measure the DNA binding kinetics. Equal volumes of protein and DNA in buffer (50 mM Tris acetate, pH 7.5, 50 mM sodium acetate, 10 mM magnesium acetate, 5 mM dithiothreitol) from separate syringes were rapidly mixed in the stopped-flow instrument at 25°C. Changes in fluorescence emission of the protein were measured using a cut-on long pass filter >348 nm (Oriel Corp., catalog number 51260) after excitation at 290 nm (1-mm slits). 2-AP fluorescence emission was measured using a cut-on filter >360 nm (WG360, Hi-Tech Scientific, serial number 273129), after excitation at 315 nm (1-5 mm slit width). About 5-10 kinetic traces for 2-AP DNAs and 20-30 for normal DNAs were routinely averaged for each experiment. The equilibrium binding and kinetic data were fit using SigmaPlot (Jandel Scientific) or KaleidaGraph (Abelbeck) softwares. Stopped-flow kinetic traces were fit using the KinTek stopped-flow kinetic program software to single or sum of exponential as in the equation, F = Σ An× exp(-kobs,nt) + C, where F is the fluorescence at time t, n is the number of exponential terms, An and kobs,n are the amplitude and the observed rate constant of the nth term, respectively, and C is the fluorescence intensity at t = 0. The error bars for kobs values shown in the Fig. 5, Fig. 6, Fig. 7 represent errors in the fit. The errors shown for the rate constants represents the mean of deviation of the reported rate constants from the values computed from the maximum and minimum kobs values.Fig. 7The 2-APΦ10 and 2-APΦ3.8 dsDNA binding as a function of [RNA polymerase]. The stopped-flow kinetic experiments described in Fig. 6, a and b, were measured at increasing [polymerase]. The first-order rate constants obtained from single exponential fit, increased linearly with increasing [polymerase] for both 2-APΦ10 (a) and 2-APΦ3.8 (b) dsDNAs. The slopes and the intercepts provided the kon and koff values of 65 ± 8 μM−1 s−1 and 3.4 ± 0.4 s−1 for 2-APΦ10 dsDNA; and 84 ± 9 μM−1 s−1, 8.7 ± 0.3 s−1 for 2-APΦ3.8 dsDNA. This translates into Kd values of 0.052 ± 0.013 and 0.104 ± 0.010 μM for 2-APΦ10 and 2-APΦ3.8 DNAs, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Stopped-flow kinetics of RNA polymerase binding to 2-APΦ10 and 2-APΦ3.8 dsDNAs. The stopped-flow kinetics of polymerase binding to 2-AP DNAs were measured at 25°C by exciting the 2-AP DNAs at 315 nm and measuring emission at >360 nm. Each kinetic trace shown is an average of 5-10 measurements. The kinetics of 2-APΦ10 (a) and 2-APΦ3.8 (b) binding under the conditions of excess polymerase (0.3 μM) over DNA (0.05 μM) fit to a single exponential. The kinetics of 2-APΦ10 (a) fit to a first-order rate constant of 19.3 ± 1.6 s−1; and 2-APΦ3.8 (b) to 28.9 ± 1.4 s−1. The kinetics of 2-APΦ10 (c) and 2-APΦ3.8 (d) binding under the conditions of excess DNA (0.3 μM) over polymerase (0.05 μM) were biphasic and fit to the sum of exponentials. The rate constants for the fast phase were comparable in value to the rate constants observed in a and b. The kinetics of 2-APΦ10 DNA (c) fit to a first-order rate constant of 17.7 ± 1.1 s−1, and the 2-APΦ3.8 DNA (d) to 35.5 ± 2.5 s−1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Studies in the literature (10Suh W.-C. Ross W. Record Jr., M.T. Science. 1993; 259: 358-361Crossref PubMed Scopus (91) Google Scholar, 11Newlands J.T. Ross W. Gosink K.K. Gourse R.L. J. Mol. Biol. 1991; 220: 569-583Crossref PubMed Scopus (74) Google Scholar, 12Ohlsen K.L. Gralla J.D. J. Biol. Chem. 1992; 267: 19813-19818Abstract Full Text PDF PubMed Google Scholar, 13Record Jr., M.T. Reznikoff W.S. Schlax P.J. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 792-820Google Scholar), mainly using E. coli RNA polymerase, have shown that binding of the RNA polymerase to the promoter DNA is a multistep process, involving formation of several closed and open polymerase-DNA binary complexes. We studied the equilibrium and kinetic interactions between T7 RNA polymerase and T7 promoters with the goal of dissecting the steps involved in the process of promoter recognition during transcription initiation. The kinetics of DNA binding were measured using stopped-flow methods. The decrease in intrinsic protein fluorescence that results upon promoter-polymerase binary complex formation was used as the probe for kinetic measurements. To investigate the kinetics of promoter opening, 2-AP containing promoter DNAs were prepared. 2-AP is a fluorescent base analog of dA that base pairs with dT. The fluorescence of 2-AP is sensitive to local changes that result from melting of dsDNA (9Fox J.J. Wempen I. Hampton A. Doerr I.L. J. Am. Chem. Soc. 1958; 80: 1669-1675Crossref Scopus (205) Google Scholar, 14Ward D.C. Reich E. Stryer L. J. Biol. Chem. 1969; 244: 1228-1237Abstract Full Text PDF PubMed Google Scholar, 15Millar D.P. Curr. Opin. Struct. Biol. 1996; 6: 322-326Crossref PubMed Scopus (118) Google Scholar). This is evident from the fluorescence spectra in Fig. 2, a and b, which shows the 2-4-fold higher fluorescence of 2-AP in the single-stranded DNA form versus the dsDNA form. Both DNA binding and DNA melting during transcription initiation should be observable in real time by following the change in fluorescence of 2-AP DNA promoters. The consensus sequence recognized by T7 RNA polymerase consists of bases in the region −17 to +6 relative to the transcription start site at +1 (16Chamberlin M. McGrath J. Waskell L. Nature. 1970; 228: 227-231Crossref PubMed Scopus (329) Google Scholar, 17Oakley J.L. Strothkamp R.E. Sarris A.H. Coleman J.E. Biochemistry. 1979; 18: 528-537Crossref PubMed Scopus (57) Google Scholar, 18Moffatt B.A. Dunn J.J. Studier F.W. J. Mol. Biol. 1984; 173: 265-269Crossref PubMed Scopus (102) Google Scholar). We have synthesized promoter DNAs, 40-base pairs in length, containing the natural Φ10 and Φ3.8 sequences from positions −21 to +19 (Fig. 1). The sequence of Φ10, a class III promoter, is absolutely conserved whereas the sequence of Φ3.8, a class II promoter, differs at several positions (−2, −11, −12, and −13) from the consensus sequence. We compare here the equilibrium and kinetic interactions of these two promoters to better understand how transcriptional efficiency is regulated at the DNA binding steps during initiation. Fluorescent promoter DNAs were chemically synthesized by incorporating 2-AP bases in place of dA bases in both the template and the nontemplate DNA strands in the region between −4 and +4 (Fig. 1). We chose to incorporate the 2-AP bases in this region because the region −6 to +2 has been shown (19Osterman H.L. Coleman J.E. Biochemistry. 1981; 20: 4884-4892Crossref PubMed Scopus (48) Google Scholar, 20Muller D.K. Martin C.T. Coleman J.E. Biochemistry. 1989; 28: 3306-3313Crossref PubMed Scopus (55) Google Scholar) to be in the single-stranded form in the open binary complex. The fluorescence of 2-AP bases at those positions should be sensitive to changes in DNA structure, such as DNA melting that occurs during initiation. Changing the dA bases to 2-AP at those positions does not affect transcription initiation. The steady state and pre-steady state kinetics of 2-mer to 19-mer RNA product formation were the same with the 2-AP-modified versus the unmodified promoters (data not shown). We have also synthesized partially dsDNA promoters that contain single-stranded template region from −5 onward as mimics of open promoter DNAs (Fig. 1). Binding of T7 RNA polymerase to 2-AP modified promoter DNAs resulted in about 6-7-fold enhancement in 2-AP-DNA fluorescence, as shown in Fig. 2, a and b. This increase in fluorescence is greater than the expected change from simply melting the duplex DNA. Therefore, at least a part of the increase in fluorescence appears to be due to the interaction of the DNA with the protein active site. The increase in 2-AP DNA fluorescence change upon binary complex formation was used to measure the equilibrium dissociation constant (Kd) of Φ10 and Φ3.8 promoters. The fluorimetric titrations were carried out at constant [DNA] and increasing [polymerase] at 25°C. The 2-AP DNA was excited at 315 nm (to minimize protein absorption) and emission was measured at 370 nm. The final equilibrium binding isotherms were obtained by subtracting the fluorescence of protein. The binding isotherms were fit to a hyperbola (Fig. 3, a and b) to calculate the apparent Kd values. The measured Kd values for Φ10 and Φ3.8 were 0.015 (±0.002) and 0.035 (±0.009) μM, respectively. The Φ10 promoter DNA therefore binds to the polymerase more strongly than the Φ3.8 promoter. The kinetic mechanism of RNA polymerase binding to DNA was investigated using the stopped-flow method. The binding of Φ10 and Φ3.8 promoter DNAs to the polymerase leads to quenching of protein tryptophan fluorescence. This intrinsic change in protein fluorescence was used to measure the kinetics of DNA binding, under conditions of excess polymerase over DNA as well as excess DNA over polymerase. Except for an extra kinetic phase under excess DNA conditions, the kinetics under the two conditions were comparable. Fig. 4 shows representative kinetic traces under excess [DNA] conditions, where 0.3 μM Φ10 DNA (Fig. 4a) or Φ3.8 DNA (Fig. 4b) was mixed with 0.05 μM polymerase. The kinetics fit best to two exponentials. To elucidate the mechanism of DNA binding and determine the bimolecular rate constant of DNA binding, the kinetics were measured as a function of increasing [DNA]. As shown in Fig. 5, a and b, the observed rate constant (kobs) of the fast phase increased linearly with increasing [DNA]. If DNA binding occurred with a simple one-step mechanism: E+D⇄koffkonEDREACTION 1 then, kobs = kon[S] + koff, where S is DNA or polymerase, E. The slope of kobs versus [DNA] provided the bimolecular rate constant, kon, and the intercept provided the dissociation rate constant, koff (21Johnson, K. A., (1992) The Enzymes, (Singman, D. S., ed), 3rd Ed., Vol. 20, pp. 1–61, Academic Press, Orlando, FL.Google Scholar). DNA binding is most likely a multistep process. Therefore, the derived kon and koff are macroscopic rate constants that describe the overall kinetics of DNA binding and dissociation and do not necessarily represent the intrinsic rate constants. The stopped-flow kinetic results show that Φ10 dsDNA promoter binds to the polymerase with a kon of 72 ± 7 μM−1 s−1 (slope), and dissociates from the binary complex with a koff of 4.0 ± 0.3 s−1 (intercept). The Φ3.8 dsDNA promoter binds to the polymerase with about 2-fold slower kon (42 ± 4 μM−1 s−1) and also dissociates faster from the complex with a 4-fold higher koff (17.7 ± 1.0 s−1). Control experiments with a dsDNA containing a non-promoter DNA sequence (Fig. 1), that is a DNA with random sequence, showed no measurable fluorescence changes confirming that the above rate constants measure specific interactions of the polymerase with the respective promoter sequences. The presence of a second slow phase in the DNA binding kinetics (Fig. 4) suggested a second step in the DNA binding mechanism (21Johnson, K. A., (1992) The Enzymes, (Singman, D. S., ed), 3rd Ed., Vol. 20, pp. 1–61, Academic Press, Orlando, FL.Google Scholar). Curiously, this slow phase was observed only when [DNA] was in excess of the polymerase. In addition, the observed rate constant of the second phase decreased with increasing [DNA] (Fig. 5, c and d). Both of these results suggest the presence of a conformational change before DNA binding, as shown in Reaction R2. The proposed conformational change is most likely a change in the polymerase, since biphasic kinetics were not observed when the polymerase concentration was in excess of the promoter DNA. E'⇄k−1k1E;E+D⇄KdEDReaction 2 According to mechanism (Reaction R2), the RNA polymerase exists in two conformations, E′ and E. The polymerase, E, is competent for DNA binding whereas polymerase, E′, needs to undergo a conformational change in order to bind DNA. k1 and k−1 represent the forward and reverse rate constants of the conformational change, and Kd is the dissociation constant of the binary complex ED. The rate constant of the second phase decreased with increasing [DNA] because of the following relationship between the observed rate constant and [DNA] (22Fersht A.R. Requena Y. J. Mol. Biol. 1971; 60: 279-290Crossref PubMed Scopus (127) Google Scholar). kobs=k1+k−1×KdKd+[DNA](Eq.1) At very low [DNA], the observed rate constant will be close to k1+ k−1, whereas at very high [DNA], the observed rate will plateau at k1. Thus, Reaction R3 predicts that the observed rate constant will decrease from the sum of the rate constants (k1+ k−1) to k1 with increasing [DNA]. This is clearly the case with the Φ3.8 promoter as shown in Fig. 5d. In case of the Φ10 promoter, the decrease is small (Fig. 5c). This is both because of the tighter Kd of Φ10 DNA relative to Φ3.8, and since the kinetics were not measured at very low [DNA]. The nature of this slow conformational change in the polymerase required for DNA binding is not known. It may represent movement of one of the polymerase domains such as the thumb region that has been postulated to be flexible and involved in DNA binding (23Sousa R. Rose J. Wang B.C. J. Mol. Biol. 1994; 244: 6-12Crossref PubMed Scopus (33) Google Scholar). Judging from the relative amplitudes of the fluorescence changes in the kinetic experiments, we estimate that E′ represents about 30% of the population of RNA polymerase. Thus, approximately 70% of the RNA polymerase is in a conformation that binds DNA with fast kinetics. The rest of the discussion in this paper is concerned with the kinetics of the fast form of the polymerase. To measure the kinetics of DNA melting, the stopped-flow experiments were repeated with the 2-AP-modified promoter DNAs. The binding of 2-AP promoter DNAs to the polymerase resulted in a time-dependent increase in 2-AP DNA fluorescence (Fig. 6). Similar to the results with the non-fluorescent DNAs, the kinetics of DNA binding at excess [polymerase] were monophasic, whereas the kinetics at excess [DNA] were multiphasic. Fig. 6, a and b, show some of the stopped-flow kinetic traces resulting from 2-APΦ10 and 2-APΦ3.8 DNA binding under the conditions of excess [polymerase] (0.3 μM) over the [DNA] (0.05 μM). Similarly, Fig. 6, c and d, show the multiphasic kinetics under conditions of excess [DNA] (0.3 μM) over [polymerase] (0.05 μM). The observed rate constants of the fast phase were comparable under both conditions. To investigate the steps involved in DNA binding and to measure their rate constants, the stopped-flow kinetics of 2-AP DNA binding were measured at increasing [polymerase]. The kinetics fit best to a single exponential and as shown in Fig. 7, a and b, the observed rate constants increased linearly as a function of [polymerase], analogous to the dependence measured by protein fluorescence change. Both protein and DNA fluorescence changes therefore appear to measure essentially the same process. The kon and koff rate constants (Table I) for 2-APΦ10 are 65 ± 8 μM−1 s−1 and 3.4 ± 0.4 s−1, respectively, and these are also comparable to those obtained from protein fluorescence changes. The kon (84 ± 9 μM−1 s−1) and koff (8.7 ± 0.3 s−1) rate constants of 2-APΦ3.8 are about 2-fold different from those of the non-fluorescent Φ3.8 DNA. The 2-AP modification does not affect the DNA binding kinetics of Φ10 promoter, but it seems to affect the kinetics of Φ3.8 promoter to a small extent. No separate phase due to DNA melting was observed. Thus, if the increase in 2-AP fluorescence measures DNA melting, this step is fast and appears to occur concomitant with DNA binding. The second slow phase observed under excess [DNA] conditions is due to the population of RNA polymerase that binds DNA with slower kinetics, since the observed rate constants of the slow phase decreased with [DNA].TABLE I.Kinetic constants of T7 RNAP binding to T7 promotersPromoterkonkoffkoff/kon = KdKdaμm−1·s−1s−1μmμmΦ1072 ± 74.0 ± 0.30.055 ± 0.0102APΦ1065 ± 83.4 ± 0.40.052 ± 0.0130.015 ± 0.002Φ10-17/40364 ± 280.02 ± 0.60.000055 ± 0.0022APΦ10-17/40329 ± 470.1 ± 1.70.0003 ± 0.006Φ3.842 ± 417.7 ± 1.00.421 ± 0.0222APΦ3.884 ± 98.7 ± 0.30.104 ± 0.0100.035 ± 0.009Φ3.8-17/40312 ± 375.6 ± 0.60.018 ± 0.00452APΦ3.8-17/40423 ± 437.7 ± 0.40.018 ± 0.001a Equilibrium constant from fluorimetric titrations. Open table in a new tab a Equilibrium constant from fluorimetric titrations. Measurement of the kinetic rate constants also provides an estimate of equilibrium constants such as Kd, (Kd = koff/kon) which provides the strength of the binary complex. Consistent with the Kd values obtained from the equilibrium fluorescence titrations (Table I), the Kd value for the 2-AP modified Φ3.8 DNA was about 2-fold higher relative to the Φ10 promoter. The equilibrium Kd values measured from the fluorimetric titrations are, however, lower than those derived from the kinetic rate constants. The different Kd values from the two methods suggest the presence of an additional step after the bimolecular DNA binding step. This step must be very slow since it was not observed by stopped-flow kinetic measurements. Additional studies need to be carried out to understand the differences in the Kd values by the two methods. To further understand the process of open binary complex formation, the kinetics of polymerase binding to partially dsDNA promoters were investigated. These promoters are double-stranded in the promoter binding region (−21 to −5), but they mimic open promoters because the template DNA in the initiation and coding regions (from −5 onward) is single-stranded. The DNA binding kinetics were measured by following both the protein fluorescence change and the 2-AP fluorescence change in the DNA. As with the fully dsDNA promoters, binding of partially dsDNA to the polymerase resulted in a decrease in protein fluorescence and an increase in 2-AP DNA fluorescence (data not shown). The kinetics of DNA binding under all conditions, excess [polymerase] or excess [DNA], fit to a single exponential. The absence of the second slow phase suggested that both E and E′ forms of the polymerase in the proposed mechanism (Reaction R1) were capable of binding the partially dsDNAs. The kon and koff values were derived from the slopes and the intercepts of the linear increases in the observed rate constant versus [DNA] or [polymerase] (Table I). Interestingly, the partially dsDNAs bind to the polymerase with much higher kon and dissociate with much lower koff values relative to the fully dsDNAs. For instance, the partially ds Φ10 DNA binds with a 5-fold higher kon, and a 30-200-fold lower koff relative to the fully dsDNA (Table I). Similarly, the partially ds Φ3.8 DNAs bind with a 5-fold higher kon, but the koff values are 2-3-fold lower than those of the fully dsDNA. We have investigated the equilibrium and kinetic interactions between T7 promoters and T7 RNA polymerase to dissect the steps in the mechanism of DNA binding during initiation of transcription. The polymerase-DNA binary complex formation was quantitated by following both the decrease in protein tryptophan fluorescence and the increase in 2-AP DNA fluorescence. Since the fluorescence of 2-AP dsDNA increases upon DNA melting, this change was used to probe the kinetics of promoter opening. The 2-AP base was introduced in place of dA bases in the region shown to be in the open form during initiation (19Osterman H.L. Coleman J.E. Biochemistry. 1981; 20: 4884-4892Crossref PubMed Scopus (48) Google Scholar, 20Muller D.K. Martin C.T. Coleman J.E. Biochemistry. 1989; 28: 3306-3313Crossref PubMed Scopus (55) Google Scholar). Replacement of dA bases with 2-AP in this region did not affect transcription initiation, since the measured steady state and pre-steady state kinetics of 2-mer to 19-mer RNA formation with 2-AP-modified DNAs as templates were comparable to those with the unmodified DNAs. We compare here the mechanism of polymerase binding to two natural T7 promoters: Φ10, a strong class III promoter, and Φ3.8, a weaker class II promoter that has base changes at positions −2, −11, −12, −13 from the consensus sequence. The base changes in class II promoters affect their efficiency of transcription, however, the regulatory mechanisms are unclear. There are several ways by which transcriptional efficiency can be regulated. A promoter may have weaker equilibrium (Kd) or unfavorable kinetic interactions with the polymerase, that is, low kon and high koff values. Thus, both kinetic and thermodynamic interactions can play a role in dictating the efficiency of transcription. Efficiency of transcription is also controlled by steps following initial DNA binding, such as open complex formation, binding of initiating nucleotides, phosphodiester bond formation, and processivity of RNA synthesis. The equilibrium interactions of Φ10 and Φ3.8 promoters with the polymerase were measured using fluorimetric titrations. The increase in fluorescence of 2-AP-modified promoters upon binding to the polymerase was used to determine the apparent Kd values. The derived Kd values indicate that T7 RNA polymerase does discriminate against Φ3.8 promoter at the DNA binding step. Φ3.8 promoter interaction with the polymerase was at least 2-fold weaker relative to the strong Φ10 promoter. Investigation of the stopped-flow kinetics of DNA binding showed that the weaker interactions of the Φ3.8 DNA were due to slower kon, the bimolecular rate constant of DNA binding, as well as faster koff, the dissociation rate constant. The Kd values calculated from the ratio of koff/kon for the various Φ3.8 promoters are also consistently weaker relative to the Φ10 promoters (see Table I). The kinetics of protein-DNA interactions (the kon and koff values) must play a significant role in promoter discrimination in vivo. The slow kon and faster koff translates into unfavorable kinetic interactions that could decrease promoter utilization. The 2-fold lower kon and the 2-fold higher koff together make Φ3.8 a weaker promoter relative to Φ10. Exactly which base change in Φ3.8 is responsible for the weaker binding cannot be determined from this study. Most likely candidates are bases at positions −11, −12, and −13 because base changes at these positions occur in T7 promoters at lower frequencies (6-18%) than changes at, say, position −2 (30%) (6Dunn J.J. Studier F.W. J. Mol. Biol. 1983; 166: 477-535Crossref PubMed Scopus (985) Google Scholar). The initial steps of DNA binding, including closed and open binary complex formation, have been studied to a large extent with E. coli RNA polymerase (13Record Jr., M.T. Reznikoff W.S. Schlax P.J. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 792-820Google Scholar). Techniques such as DNA footprinting and nitrocellulose membrane binding have been used to measure the kinetics of DNA binding. Similarly, sensitivity of open single-stranded DNA to KMnO4 (24Hayatsu H. Ukita T. Biochem. Biophys. Res. Commun. 1967; 29: 556-561Crossref PubMed Scopus (117) Google Scholar, 25Kainz M. Roberts J. Science. 1992; 255: 838-841Crossref PubMed Scopus (70) Google Scholar), and nitrocellulose filter binding (26Roe J.H. Burgess R.R. Record Jr., M.T. J. Mol. Biol. 1985; 184: 441-453Crossref PubMed Scopus (143) Google Scholar) and polyacrylamide gel-retardation assay with heparin chase (27Zhang X. Reeder T. Schleif R. J. Mol. Biol. 1996; 258: 14-24Crossref PubMed Scopus (52) Google Scholar, 28Wellington S.R. Spiegelman G.B. J. Biol. Chem. 1993; 268: 7205-7214Abstract Full Text PDF PubMed Google Scholar) have been used to probe open complex formation steps. All of these methods are manual and cannot measure transient complexes formed with rapid kinetics. The stopped-flow method, especially with promoter DNAs containing the fluorescent base 2-AP, is ideal for measuring the kinetics of DNA binding as well as the kinetics of promoter opening in real time. The fluorescence of 2-AP is sensitive to changes in the state of the DNA. Thus, stopped-flow kinetic studies with promoter DNAs containing 2-AP bases placed in the initiation region allow measurement of local conformational changes, such as DNA melting, that occur during initiation. Additionally, these experiments provide both kinetic and equilibrium information necessary to elucidate the detailed mechanism of DNA binding during transcription. To dissect the various steps in the mechanism of promoter binding, the kinetics of DNA binding were measured by following both the changes in the fluorescence of protein and the 2-AP DNA. These experiments indicated that both protein and DNA fluorescence changes measured the same bimolecular process of DNA binding to the protein. No distinct kinetic phase due to promoter opening was observed with the fluorescent 2-AP DNAs. Stopped-flow study of both protein and DNA fluorescence changes provided the kon and koff rate constants that describe the overall rate of binary complex formation and dissociation. Comparison of the kinetic constants for different promoters shows that the strength of binary complex is determined by both the kon and the koff values (Table I). Partially dsDNA promoters bind to the polymerase with the highest affinity. The strong binding of partially dsDNAs is due to both a higher kon (5-10-fold) and a lower koff (2-200 fold) compared to the fully dsDNAs. Furthermore, it is also seen, that the ds Φ10 promoter binds to the polymerase strongly, and with a higher kon (about 2-fold) and a lower koff (about 2-fold) relative to the weaker ds Φ3.8 promoter. The lower koff values indicate that the binary complex is kinetically stable. Thus, the partially dsDNAs form the most kinetically stable binary complexes whereas the Φ3.8 dsDNAs the least. The lower than diffusion limited kon and the differences in kon values between various promoter DNAs suggest that DNA binding occurs by a two-step mechanism, E+D⇄K1EDa⇄k2k−2EDbReaction 3 The first step in the above mechanism represents the diffusion-controlled binding of the DNA to the polymerase to form EDa, with an equilibrium association constant K1. The interactions of the DNA with polymerase in this complex are weak, and the free polymerase, DNA, and the EDa species are in rapid equilibrium (that is, the rates of binding and dissociation are faster relative to k2 and k−2). Therefore, although there are two steps in the DNA binding mechanism, due to fast formation of EDa, the stopped-flow kinetics showed only one phase. The second step leading to the formation of EDb is a conformational change, wherein the interactions between the polymerase and the DNA are more stable. If we compare the above mechanism to that proposed for DNA binding to E. coli RNA polymerase (13Record Jr., M.T. Reznikoff W.S. Schlax P.J. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 792-820Google Scholar), the EDa complex would be analogous to one of the closed binary complexes, and the EDb species may be analogous to one of the open complexes. Since the stopped-flow kinetics of DNA binding measured by protein or DNA fluorescence changes were identical, the isomerization step may be global. In other words, both polymerase and DNA may change their conformation concomitantly during the isomerization step leading to formation of EDb. Studies with partially dsDNA promoters (both 2-AP and unmodified DNAs) provide clues as to the nature of this second step. Since the partially dsDNAs are already open, we did not expect the 2-AP fluorescence to change. However, the 2-AP fluorescence in the partially dsDNAs increased in an analogous manner as in the fully dsDNAs. These results can be explained in two ways. The 2-AP fluorescence increase may be due to a conformational change that occurs in both fully double-stranded and partially dsDNAs such as DNA twisting or DNA bending, distortions which could lead to promoter opening (29Travers A.A. Crit. Rev. Biochem. 1987; 22: 181-219Crossref PubMed Scopus (56) Google Scholar). Thus, DNA bending or twisting may represent the second step in mechanism (Reaction R3), and the resulting EDb complex in the fully dsDNA promoters may be an intermediate open binary complex. Alternatively, the increase in 2-AP fluorescence may simply be due to the change in the protein conformation which may result in changes in the local environment around the DNA. According to the proposed mechanism (Reaction R3), the bimolecular rate constant, kon listed in Table I is equal to K1k2 and the measured dissociation rate constant, koff is equal to k−2. The lower-than-diffusion-limited kon values for the fully dsDNA promoters can result both from a weaker EDa complex or a slower conformational change (k2). The Φ3.8 promoter, which has a lower kon, therefore forms a weaker EDa complex or undergoes a slower conformational change, or both, relative to the Φ10 promoter. Since the partially dsDNAs are already open, they do not have to go through a “closed” complex, and therefore the kon values of partially dsDNAs are higher and close to diffusion-limited. The above two-step mechanism of DNA binding predicts that the observed rate constants should saturate at k2+ k−2 at high DNA or polymerase concentrations. Since no saturation was observed in our measurements up to rate constant of 100 s−1, the second step in the above mechanism must be fast (>100 s−1) for both the Φ10 and Φ3.8 promoters. Relative to the rates of transcription initiation, 2In T7 RNA polymerase, transcription initiation occurs at rate constants ranging from 1 to 5 s−1 (Y. Jia and S. S. Patel, unpublished results). EDb, the intermediate open binary complex, is formed with fast kinetics that cannot limit transcription initiation. This does not mean that regulation cannot occur at the DNA binding step. Although promoter opening is not rate-limiting, the equilibrium constants of DNA binding and isomerization steps can modulate the efficiency of transcription initiation. For instance, the faster koff (or k−2) of Φ3.8 makes the isomerization of EDa to EDb to occur with an unfavorable equilibrium relative to the Φ10 promoter. Thus, efficiency of transcription initiation is modulated kinetically by the macroscopic rate constants kon and koff, and thermodynamically by the equilibrium constants (K1 and k2/k−2) of the two steps during DNA binding." @default.
• W2056588849 created "2016-06-24" @default.
• W2056588849 creator A5015815507 @default.
• W2056588849 creator A5034388856 @default.
• W2056588849 creator A5042153355 @default.
• W2056588849 date "1996-11-01" @default.
• W2056588849 modified "2023-09-28" @default.
• W2056588849 title "Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes" @default.
• W2056588849 cites W1500658881 @default.
• W2056588849 cites W1506711440 @default.
• W2056588849 cites W1518712848 @default.
• W2056588849 cites W1531677604 @default.
• W2056588849 cites W1560186970 @default.
• W2056588849 cites W1579716589 @default.
• W2056588849 cites W1594732601 @default.
• W2056588849 cites W1642267050 @default.
• W2056588849 cites W1805448417 @default.
• W2056588849 cites W1966158292 @default.
• W2056588849 cites W1976374524 @default.
• W2056588849 cites W2002841189 @default.
• W2056588849 cites W2012174863 @default.
• W2056588849 cites W2021968860 @default.
• W2056588849 cites W2022890528 @default.
• W2056588849 cites W2027930327 @default.
• W2056588849 cites W2057263492 @default.
• W2056588849 cites W2065861374 @default.
• W2056588849 cites W2069525245 @default.
• W2056588849 cites W2075672090 @default.
• W2056588849 cites W2085833379 @default.
• W2056588849 cites W2091596891 @default.
• W2056588849 cites W2098514662 @default.
• W2056588849 cites W2103947911 @default.
• W2056588849 cites W2166172853 @default.
• W2056588849 cites W2200851602 @default.
• W2056588849 cites W4212901177 @default.
• W2056588849 doi "https://doi.org/10.1074/jbc.271.48.30451" @default.
• W2056588849 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8940010" @default.
• W2056588849 hasPublicationYear "1996" @default.
• W2056588849 type Work @default.
• W2056588849 sameAs 2056588849 @default.
• W2056588849 citedByCount "93" @default.
• W2056588849 countsByYear W20565888492012 @default.
• W2056588849 countsByYear W20565888492013 @default.
• W2056588849 countsByYear W20565888492014 @default.
• W2056588849 countsByYear W20565888492015 @default.
• W2056588849 countsByYear W20565888492016 @default.
• W2056588849 countsByYear W20565888492017 @default.
• W2056588849 countsByYear W20565888492018 @default.
• W2056588849 countsByYear W20565888492019 @default.
• W2056588849 countsByYear W20565888492021 @default.
• W2056588849 countsByYear W20565888492023 @default.
• W2056588849 crossrefType "journal-article" @default.
• W2056588849 hasAuthorship W2056588849A5015815507 @default.
• W2056588849 hasAuthorship W2056588849A5034388856 @default.
• W2056588849 hasAuthorship W2056588849A5042153355 @default.
• W2056588849 hasBestOaLocation W20565888491 @default.
• W2056588849 hasConcept C104317684 @default.
• W2056588849 hasConcept C121332964 @default.
• W2056588849 hasConcept C12554922 @default.
• W2056588849 hasConcept C135889238 @default.
• W2056588849 hasConcept C181199279 @default.
• W2056588849 hasConcept C185592680 @default.
• W2056588849 hasConcept C2776449523 @default.
• W2056588849 hasConcept C55493867 @default.
• W2056588849 hasConcept C62520636 @default.
• W2056588849 hasConcept C67705224 @default.
• W2056588849 hasConcept C82381507 @default.
• W2056588849 hasConcept C86803240 @default.
• W2056588849 hasConcept C91881484 @default.
• W2056588849 hasConceptScore W2056588849C104317684 @default.
• W2056588849 hasConceptScore W2056588849C121332964 @default.
• W2056588849 hasConceptScore W2056588849C12554922 @default.
• W2056588849 hasConceptScore W2056588849C135889238 @default.
• W2056588849 hasConceptScore W2056588849C181199279 @default.
• W2056588849 hasConceptScore W2056588849C185592680 @default.
• W2056588849 hasConceptScore W2056588849C2776449523 @default.
• W2056588849 hasConceptScore W2056588849C55493867 @default.
• W2056588849 hasConceptScore W2056588849C62520636 @default.
• W2056588849 hasConceptScore W2056588849C67705224 @default.
• W2056588849 hasConceptScore W2056588849C82381507 @default.
• W2056588849 hasConceptScore W2056588849C86803240 @default.
• W2056588849 hasConceptScore W2056588849C91881484 @default.
• W2056588849 hasIssue "48" @default.
• W2056588849 hasLocation W20565888491 @default.
• W2056588849 hasOpenAccess W2056588849 @default.
• W2056588849 hasPrimaryLocation W20565888491 @default.
• W2056588849 hasRelatedWork W1484706750 @default.
• W2056588849 hasRelatedWork W1493626684 @default.
• W2056588849 hasRelatedWork W1659766963 @default.
• W2056588849 hasRelatedWork W1989149131 @default.
• W2056588849 hasRelatedWork W2005525247 @default.
• W2056588849 hasRelatedWork W2063381905 @default.
• W2056588849 hasRelatedWork W2131907796 @default.
• W2056588849 hasRelatedWork W2136461133 @default.
• W2056588849 hasRelatedWork W2306656032 @default.
• W2056588849 hasRelatedWork W3025511956 @default.
• W2056588849 hasVolume "271" @default.
• W2056588849 isParatext "false" @default.

• next