PURIFICATION AND CHARACTERIZATION OF A SOLUBLE DNA-DEPENDENT CHLOROPLAST RNA-POLYMERASE FROM PISUM-SATIVUM

A chloroplast DNA-dependent RNA polymerase has been purified 43 000-fo ld from pea leaves. The purification procedure includes: 100 000 x g c entrifugation, DE-52 ion-exchange chromatography, ammonium sulfate pre cipitation, Sephacryl-300 gel filtration and two FPLC Mono-Q columns. The native molecular mass of the purified enzyme is approximate to 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 d uring SDS-PAGE. The enzyme is completely dependent on an exogenous DNA template for activity. The 110 kDa polypeptide binds nucleoside triph osphates. 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 GT P and the V-max is 200 pmol/min per mg. The optimum conditions for max imum enzyme activity are 2 mM KCl, 15 mM MgCl2 (or MnCl2), 40 degrees C, and pH 7.9. Enzyme activity is inhibited by added (NH4)(2)SO4, tage titoxin and heparin. The purified soluble RNA polymerase selectively I nitiates transcription from chloroplast mRNA promoters of higher plant and the Euglena chloroplast tRNAPhe promoter. (C) 1998 Published by E lsevier Science Ireland Ltd. All rights reserved.