Abstract
A chloroplast DNA-dependent RNA polymerase has been purified 43 000-fold from pea leaves. The purification procedure includes: 100 000× g centrifugation, DE-52 ion-exchange chromatography, ammonium sulfate precipitation, Sephacryl-300 gel filtration and two FPLC Mono-Q columns. The native molecular mass of the purified enzyme is ≈620 000 based on gel filtration chromatography, and 669 000 based on non-denaturing gel electrophoresis. The purified enzyme is separated into ten polypeptides of 120, 110, 95, 84, 81, 75, 54, 51, 42, and 35 kDa during SDS-PAGE. The enzyme is completely dependent on an exogenous DNA template for activity. The 110 kDa polypeptide binds nucleoside triphosphates. The 42 kDa polypeptide cross-reacts with antiserum raised to the plastid encoded rpoA gene product. The K m value is 0.1 mM for GTP and the V max is 200 pmol/min per mg. The optimum conditions for maximum enzyme activity are 2 mM KCl, 15 mM MgCl 2 (or MnCl 2), 40°C, and pH 7.9. Enzyme activity is inhibited by added (NH 4) 2SO 4, tagetitoxin and heparin. The purified soluble RNA polymerase selectively initiates transcription from chloroplast mRNA promoters of higher plant and the Euglena chloroplast tRNA Phe promoter.
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