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

*† ‡ Summary We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the a subunit containing a C-terminal 12 x histidine tag using heparin and Ni2þ 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 a, b, b¢, b¢¢ 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.