Sequence Characteristics Required for Cooperative Binding and Efficient in Vivo Titration of the Replication Initiator Protein DnaA in E. coli

Flemming G. Hansen, Bjarke Bak Christensen, Tove Atlung

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    Resumé

    Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TTA/TTNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional DnaA boxes around the promoter proximal R5 DnaA box were identified and shown by mutational analysis to be necessary for the cooperative binding of DnaA required for titration. These four DnaA boxes are located in the same orientation and with a spacing of two or three base-pairs. The cooperative binding was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp region containing the R5 DnaA box, while repression of mioC transcription by DnaA, which is dependent on the R5 DnaA box, was independent of the two DnaA boxes downstream of R5. Repression was also independent of the spacing between the two upstream DnaA boxes and the promoter as long as a DnaA box was located less than 20 bp upstream of the - 35 sequence. Thus, the architectural requirements for titration and for repression of transcription are different. A new set of rules for identifying efficiently titrating DnaA box regions was formulated and used to analyse sequences for which good titration data are available. © 2007 Elsevier B.V. All rights reserved
    OriginalsprogEngelsk
    TidsskriftJournal of Molecular Biology
    Vol/bind367
    Udgave nummer4
    Sider (fra-til)942-952
    Antal sider11
    ISSN0022-2836
    DOI
    StatusUdgivet - 2007

    Citer dette

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    title = "Sequence Characteristics Required for Cooperative Binding and Efficient in Vivo Titration of the Replication Initiator Protein DnaA in E. coli",
    abstract = "Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TTA/TTNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional DnaA boxes around the promoter proximal R5 DnaA box were identified and shown by mutational analysis to be necessary for the cooperative binding of DnaA required for titration. These four DnaA boxes are located in the same orientation and with a spacing of two or three base-pairs. The cooperative binding was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp region containing the R5 DnaA box, while repression of mioC transcription by DnaA, which is dependent on the R5 DnaA box, was independent of the two DnaA boxes downstream of R5. Repression was also independent of the spacing between the two upstream DnaA boxes and the promoter as long as a DnaA box was located less than 20 bp upstream of the - 35 sequence. Thus, the architectural requirements for titration and for repression of transcription are different. A new set of rules for identifying efficiently titrating DnaA box regions was formulated and used to analyse sequences for which good titration data are available. {\circledC} 2007 Elsevier B.V. All rights reserved",
    keywords = "DnaA protein, DnaA box spacing, mioC promoter, in vivo titration, repression of mioC transcription",
    author = "Hansen, {Flemming G.} and {Bak Christensen}, Bjarke and Tove Atlung",
    year = "2007",
    doi = "10.1016/j.jmb.2007.01.056",
    language = "English",
    volume = "367",
    pages = "942--952",
    journal = "Journal of Molecular Biology",
    issn = "0022-2836",
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    Sequence Characteristics Required for Cooperative Binding and Efficient in Vivo Titration of the Replication Initiator Protein DnaA in E. coli. / Hansen, Flemming G.; Bak Christensen, Bjarke; Atlung, Tove.

    I: Journal of Molecular Biology, Bind 367, Nr. 4, 2007, s. 942-952.

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    TY - JOUR

    T1 - Sequence Characteristics Required for Cooperative Binding and Efficient in Vivo Titration of the Replication Initiator Protein DnaA in E. coli

    AU - Hansen, Flemming G.

    AU - Bak Christensen, Bjarke

    AU - Atlung, Tove

    PY - 2007

    Y1 - 2007

    N2 - Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TTA/TTNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional DnaA boxes around the promoter proximal R5 DnaA box were identified and shown by mutational analysis to be necessary for the cooperative binding of DnaA required for titration. These four DnaA boxes are located in the same orientation and with a spacing of two or three base-pairs. The cooperative binding was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp region containing the R5 DnaA box, while repression of mioC transcription by DnaA, which is dependent on the R5 DnaA box, was independent of the two DnaA boxes downstream of R5. Repression was also independent of the spacing between the two upstream DnaA boxes and the promoter as long as a DnaA box was located less than 20 bp upstream of the - 35 sequence. Thus, the architectural requirements for titration and for repression of transcription are different. A new set of rules for identifying efficiently titrating DnaA box regions was formulated and used to analyse sequences for which good titration data are available. © 2007 Elsevier B.V. All rights reserved

    AB - Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TTA/TTNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional DnaA boxes around the promoter proximal R5 DnaA box were identified and shown by mutational analysis to be necessary for the cooperative binding of DnaA required for titration. These four DnaA boxes are located in the same orientation and with a spacing of two or three base-pairs. The cooperative binding was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp region containing the R5 DnaA box, while repression of mioC transcription by DnaA, which is dependent on the R5 DnaA box, was independent of the two DnaA boxes downstream of R5. Repression was also independent of the spacing between the two upstream DnaA boxes and the promoter as long as a DnaA box was located less than 20 bp upstream of the - 35 sequence. Thus, the architectural requirements for titration and for repression of transcription are different. A new set of rules for identifying efficiently titrating DnaA box regions was formulated and used to analyse sequences for which good titration data are available. © 2007 Elsevier B.V. All rights reserved

    KW - DnaA protein

    KW - DnaA box spacing

    KW - mioC promoter

    KW - in vivo titration

    KW - repression of mioC transcription

    U2 - 10.1016/j.jmb.2007.01.056

    DO - 10.1016/j.jmb.2007.01.056

    M3 - Journal article

    VL - 367

    SP - 942

    EP - 952

    JO - Journal of Molecular Biology

    JF - Journal of Molecular Biology

    SN - 0022-2836

    IS - 4

    ER -