Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme.
We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha 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 alpha, beta, beta', beta" 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.
Suzuki
Jon Y
JY
Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
Ytterberg
A Jimmy
AJ
Beardslee
Thomas A
TA
Allison
Lori A
LA
Wijk
Klaas Jan
KJ
Maliga
Pal
P
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification; D018087:Plastids / Q000201:enzymology*; D021122:Protein Subunits / Q000302:isolation & purification; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure
D010940:Plant Proteins; D021122:Protein Subunits; D012321:DNA-Directed RNA Polymerases