Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme

Authors

  • Jon Y. Suzuki,

    1. Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA,
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    • Present address: USDA/ARS/PWA,US Pacific Basin Agricultural Research Center, PO Box 4459, 920 Stainback Highway, Hilo, HI 96720, USA.

  • A. Jimmy Ytterberg,

    1. Department of Plant Biology, Emerson Hall 332, Tower Road, Cornell University, Ithaca, NY 14853, USA,
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  • Thomas A. Beardslee,

    1. Department of Biochemistry, N258 Beadle Center, University of Nebraska, Lincoln, NE 68588-0664, USA, and
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    • Present address: Invitrogen Corporation, 1600 Faraday Ave., Carlsbad, CA 92008, USA.

  • Lori A. Allison,

    1. Department of Biochemistry, N258 Beadle Center, University of Nebraska, Lincoln, NE 68588-0664, USA, and
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  • Klaas Jan van Wijk,

    1. Department of Plant Biology, Emerson Hall 332, Tower Road, Cornell University, Ithaca, NY 14853, USA,
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  • Pal Maliga

    Corresponding author
    1. Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA,
    2. Department of Plant Biology, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ, 08901, USA
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(fax 732 445 5735; e-mail maliga@waksman.rutgers.edu).

Summary

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the α subunit containing a C-terminal 12 x histidine tag using heparin and Ni2+ chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core α, β, β′, β′′ subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.

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