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2002 | 49 | 3 | 659-669
Article title

Effect of An tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

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In the open transcription complex (RPo), Escherichia coli RNA polymerase s70 and α subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of An DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i) A5 within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii) A5 (Pg) or A8 (Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant ka of RPo formation is: (i) by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K1, of the initial closed complex; and (ii) several-fold smaller at Pg and Pm owing to a strongly decreased value of K1. For Pm, the latter parameter was found to be dependent exponentially on four Mg2+ ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A8·T8 fragment of B'-DNA forms a smaller number of ionic contacts with the α subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase α and s70 subunits with the proximal UP subsite and the -35 region of promoters.
Physical description
  • Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
  • Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
  • Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
  • Bertrand-Burggraf E, Lefevre JF, Daune M. (1984) A new experimental approach for studying the association between RNA polymerase and the tet promoter of pBR322. Nucleic Acids Res.; 12: 1697-706.
  • Buc H, McClureWR. (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: 2712-23.
  • Burgess RR, Jendrisak JJ. (1975) A procedure for the rapid large-scale purification of Escherichia coli DNA-dependent RNA polymerase involving polymin P precipitation and DNA-cellulose chromotography. Biochemistry.; 14: 4634-8.
  • 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: 540-68.
  • Dombroski AJ, Walter WA, Record Jr MT, Siegele DA, Gross CA. (1992) Polypeptides containing highly conserved regions of transcription initiation factor s70 exhibit specificity of binding to promoter DNA. Cell.; 70: 501-12.
  • Estrem ST, Ross W, Gaal T, Chen ZW, Niu W, Ebright RH. (1999) Bacterial promoter architecture: subsite structure of UP elements and interaction with the carboxy-terminal domain of the RNA polymerase a subunit. Genes Dev.; 13: 2134-47.
  • Helmann JD, deHaseth PL. (1999) Protein-nucleic acid interactions during open complex formation investigated by systematic alteration of the protein and DNA binding partners. Biochemistry.; 38: 5959-67.
  • Hertz GZ, Stormo GD. (1996) Escherichia coli promoter sequences: analysis and prediction. Methods Enzymol.; 273: 30-42.
  • Heyduk E, Baichoo N, Heyduk T. (2001) Interaction of the a-subunit of Escherichia coli RNA polymerase with DNA. J Biol Chem.; 276: 44598-603.
  • Jeon YH, Negishi T, Shirakava M, Yamazaki T, Fujita N, Ishihama A, Kyogoku N. (1995) Solution structure of the activator contact domain of the RNA polymerase a subunit. Science.; 270: 1495-7.
  • Kolasa IK. (2001) Influence of An·Tn DNA bending sequences on Escherichia coli promoter strength in vitro. Ph.D. Thesis. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland (in Polish).
  • Kolasa IK, Lozinski T, Wierzchowski KL. (2001) Mg2+ does not induce isomerization of the open transcription complex of Escherichia coli RNA polymerase at the model Pa promoter bearing consensus -10 and -35 hexamers. Acta Biochim Polon.; 48: 985-94.
  • Leirmo S, Record MT Jr. (1990) Structural thermodynamic and kinetic studies of the interaction of Es70 RNA polymerase with promoter DNA. In Nucleic acids and molecular biology. Eckstein F, Lilley DMJ. eds, vol 4, pp 123-151. Springer-Verlag, Heidelberg.
  • Lozinski T, Wierzchowski KL. (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: 265-80.
  • Lozinski T, Markiewicz WT, Wykrzykiewicz TK, Wierzchowski KL. (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: 3855-63.
  • Lozinski T, Adrych-Rozek K, Markiewicz WT, Wierzchowski KL. (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: 2947-53.
  • MacDonald D, Herbert K, Zhang X, Polgruto T, Lu P. (2001) Solution structure of an A-tract DNA bend. J Mol Biol.; 306: 1081-98.
  • Meng W, Belyaeva T, Savery NJ, Busby SJW, Ross WE, Gaal T, Gourse RL, Thomas MS. (2001) UP element-dependent transcription at the Escherichia coli rrnBP1 promoter: positional requirements and role of the RNA polymerase a subunit linker. Nucleic Acids Res.; 29: 4166-78.
  • Mekler V, Kortkhonija K, Mukhopadhyay J, Knight J, Revyakin A, Kapanidis AN, Niu W, Ebright YW, Levy R, Ebright RH. (2002) Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell.; 108: 599-614.
  • Misra VK, Draper DE. (1999) The interpretation of Mg2+ binding isotherms for nucleic acids using Poisson-Boltzmann theory. J Mol Biol.; 294: 1135-47.
  • Naryshkin N, Revyakin A, Kim Y, Mekler V, Ebright RH. (2000) Structural organization of the RNA polymerase-promotor open complex. Cell.; 101: 601-11.
  • Nelson HCM, Finch JT, Luisi BF, Klug A. (1987) The structure of an oligo(dA),oligo(dT) tract and its biological implications. Nature (London).; 330: 221-6.
  • Record MT Jr, deHaseth PL, Lohman TM. (1977) Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction. Biochemistry.; 16: 4791-6.
  • Roe J-H, Burgess RR, Record MT Jr. (1984) Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lambdaPR promoter. J Mol Biol,; 176: 495-521.
  • Roe JH, Burgess RR, Record MT Jr. (1985) Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambdaPR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening. J Mol Biol.; 184: 441-53.
  • Rosenberg S, Kadesch TR, Chamberlin MJ. (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.
  • Ross W, Aiyar SE, Gourse RL. (1998) Escherichia coli promoters with UP elements of different strength: modular structure of bacterial promoters. J Bacteriol.; 180: 5375-83.
  • Ross W, Ernst A, Gourse RL. (2001) Fine structure of E. coli RNA polymerase-promoter interactions: a subunit binding to the UP element minor groove. Genes Dev.; 15: 491-506.
  • Shao X, Grishin NV. (2000) Common fold in helix-hairpin-helix proteins. Nucleic Acids Res.; 28: 2643-50.
  • Siegele DA, Hu JC, Walter WA, Gross CA. (1989) Altered promoter recognition by mutant forms of the s70 subunit of Escherichia coli RNA polymerase. J Mol Biol.; 206: 591-603.
  • Strainic MG Jr, Sullivan JJ, Vevelis A, deHaseth PL. (1998) Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open complex formation and promoter clearance. Biochemistry.; 37: 18074-80.
  • Suh WC, Leirmo S, Record MT Jr. (1992) Role of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambdaPR promoter: kinetic evidence for a second open complex requiring Mg2+. Biochemistry.; 31: 7815-25.
  • Tsodikov OV, Record MT 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 Es70 RNA polymerase and lambdaPR promoter DNA. Biophys J.; 76: 1320-9.
  • Yarbrough LR, Schlageck JG, Baughman M. (1979) Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases. J Biol Chem.; 254: 12069-73.
  • Yasuno K, Yamazaki T, Tanaka Y, Kodama TS, Matsugami A, Katahira M, Ishihama A, Kyogoku Y. (2001) Interaction of the C-terminal domain of the E. coli RNA polymerase a subunit with the UP element: recognizing the backbone structure in the minor groove surface. J Mol Biol.; 306: 213-25.
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