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Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme
Author(s) -
Suzuki Jon Y.,
Jimmy Ytterberg A.,
Beardslee Thomas A.,
Allison Lori A.,
Wijk Klaas Jan,
Maliga Pal
Publication year - 2004
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2004.02195.x
Subject(s) - plastid , rna polymerase , biology , specificity factor , sigma factor , protein subunit , recombinant dna , microbiology and biotechnology , biochemistry , affinity chromatography , polymerase , rna , chloroplast , gene , enzyme
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 Ni 2+ 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.