Mg 2+ Does not induce isomerization of the open transcription complex Escherichia coli RNA polymerase at the model Pα promoter bearing consensus -10 and -35 hexamers.
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The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pα promoter bearing consensus -10 and -35 recognition hexamers were studied in vitro. Previously, this promoter was used as a control one in studies on the effect of DNA bending by An·Tn sequences on transcription initiation and shown to be fully functional in E. coli (Łoziński et al., 1991, Nucleic Acids Res. 19, 2947; Łoziński & Wierzchowski, 1996, Acta Biochim. Polon. 43, 265). The data now obtained demonstrate that the mechanism of Pα-RPo formation and dissociation conforms to the three-step reaction model: bind-nucleate-melt, commonly accepted for natural promoters. Measurements of the dissociation rate constant of Pα -RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg≈ 4, being bound to the RPo in the course of renaturation of the melted DNA region. This number was found constant in the temperature range of 25-37°C, which indicates that under these conditions the complex remaines fully open. This observation, taken together with the recent evidence from independent of the presence of Mg2+ ions (Łoziński & Wierzchowski, 2001, Acta KMnO4 footprinting studies that the length of the melted region in Pα-RPo at 37°C is Biochim. Polon. 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the λPR-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).
- Bertrand-Burggraf, E., Lefévre, J.F. & Daune, M. (1984) A new experimental approach for studying the association between RNA polymerase and the tet promoter of pBR322. Nucleic Acids Res. 12, 1697-1706.
- Buc, H. & McClure, W.R. (1985) Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps. Biochemistry 24, 2712-2723.
- Breslauer, K.J., Frank, R., Blocker, H. & Marky, L.A. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750.
- Burgess, R.R. & Jendrisak, J.J. (1975) A procedure for the rapid, large-scale purification of Escherichia coli DNA-dependent RNA polymerase involving polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14, 4634-4638.
- Chamberlin, M., Kingston, R., Gilman, M., Wiggs, J. & deVera, A. (1983) Isolation of bacterial and bacteriophage RNA polymerases and their use in synthesis of RNA in vitro. Methods Enzymol. 101, 540-568.
- Chen, Y.-F. & Helmann, J.D. (1997) DNA melting at the Bacillus subtilis flagellin promoter nucleates near -10 and expands unidirectionally. J. Mol. Biol. 267, 47-59.
- DeHaseth, P.L., Zupancic, M.L. & Record, M.T., Jr. (1998) RNA polymerase-promoter interactions: The comings and goings of RNA polymerase. J. Bacteriol. 180, 3019-3025.
- Duval-Valentin, G. & Ehrlich, R. (1987) Dynamic and structural characterization of multiple steps during complex formation between E. coli RNA polymerase and tetR promoter from pSC101. Nucleic Acids Res. 15, 575-594.
- Helmann, J.D. & deHaseth, P.L. (1999) Protein-nucleic acid interactions during open complex formation investigated by systematic alteration of the protein and DNA binding partners. Biochemistry 38, 5959-5967.
- Kolasa, I.K. (2001) Wplyw sekwencji An · Tn zaginajacych DNA na moc promotora Escherichia coli in vitro. Ph.D. Thesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland (in Polish).
- Leirmo, S. & Record, M.T., Jr. (1990) Structural, thermodynamic and kinetic studies of the interaction of E70 RNA polymerase with promoter DNA; in Nucleic Acids and Molecular Biology (Eckstein, F. & Lilley, D.M.J., eds.) vol 4, pp. 123-151, Springer-Verlag, Heidelberg.
- Łoziński, T., Markiewicz, W.T., Wykrzykiewicz, T.K. & Wierzchowski, K.L. (1989) Effect of the sequence-dependent structure of the 17 bp AT spacer on the strength of consensus-like E. coli promoters in vivo. Nucleic Acids Res. 17, 3855-3863.
- Łoziński, T., Adrych-Roek, K., Markiewicz, W.T. & Wierzchowski, K.L. (1991) Effect of DNA bending in various regions of the consensus-like Escherichia coli promoter on its strength in vivo and structure of the open complex in vitro. Nucleic Acids Res. 19, 2947-2953.
- Łoziński, T. & Wierzchowski,K.L. (1996) Effect of reversed orientation and length of An · Tn DNA bending sequences in the -35 and spacer domains of a consensus-like Escherichia coli promoter on its strength in vivo and gross structure of the open complex in vitro. Acta Biochim. Polon. 43, 265-280.
- Łoziński, T. & Wierzchowski, K.L. (2001) Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic P a promoter. A quantitative KMnO4 footprinting study. Acta Biochim. Polon. 48, 495-510.
- Misra, V.K. & Draper, D.E. (1999) The interpretation of Mg2+ binding isotherms for nucleic acids using Poisson-Boltzmann theory. J. Mol. Biol. 294, 1135-1147.
- Record, M.T., Jr., deHaseth, P.L. & Lohman, T.M. (1977) Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction. Biochemistry 16, 4791-4796.
- Roe, J.-H., Burgess, R.R. & Record, M.T., Jr. (1984) Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the λ PR promoter. J. Mol. Biol. 176, 495-521.
- Roe, J.H., Burgess, R.R. & Record, M.T., Jr. (1985) Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-λ PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening. J. Mol. Biol. 184, 441-453.
- Rosenberg, S., Kadesch, T.R. & Chamberlin, M.J. (1982) Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA. Measurements of the rate of open complex formation at T7 promoter A1. J. Mol. Biol. 155, 31-51.
- Strainic, M.G., Jr., Sullivan, J.J., Vevelis, A. & deHaseth, P.L. (1998) Promoter recognition by Escherichia coli RNA polymerase: Effects of the UP element on open complex formation and promoter clearance. Biochemistry 37, 18074-18080.
- Suh, W.C., Leirmo, S. & Record, M.T., Jr. (1992) Role of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the λPR promoter: Kinetic evidence for a second open complex requiring Mg2+. Biochemistry 31, 7815-7825.
- Suh, W.C., Ross, W. & Record, M.T., Jr. (1993) Two open complexes and a requirement for Mg2+ to open the λPR transcription start site. Science 259, 358-361.
- Tsodikov, O.V. & Record, M.T., Jr. (1999) General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime: Application to kinetics of open complex formation between Eσ70 RNA polymerase and λPR promoter DNA. Biophys J. 76, 1320-1329.
- Yarbrough, L.R., Schlageck, J.G. & Baughman, M. (1979) Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases. J. Biol. Chem. 254, 12069-12073.
- Zaychikov, E., Denissova, L., Meier, T., Gotte, M. & Heumann, H. (1997) Influence of Mg2+ and temperature on formation of the transcription bubble. J. Biol. Chem. 272, 2259-2267.
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