Properties and Characterization of a Spinach Chloroplast RNA Polymerase Isolated from a Transcriptionally Active DNA‐Protein Complex

Abstract

A chloroplast RNA polymerase has been isolated from a transcriptionally active spinach plastid DNA‐protein complex. The properties of the complex and of the reconstituted system have been compared. The crude enzyme, is at least sevenfold less active when compared with the complex. RNA synthesis by the reconstituted system is sensitive to high ionic strength and heparin, contrarily to RNA synthesis by the chloroplast DNA‐protein complex. On the other hand, rifampicin has no inhibitory effect whatever on the transcriptional system used. The RNA polymerase isolated is more efficient with denatured DNA than with double‐stranded DNA and the best template is chloroplast DNA. The crude RNA polymerase isolated migrates in a peak of 11 S in glycerol gradient centrifugation and is located in a single band in non‐denaturing polyacrylamide gel electrophoresis. About 30 polypeptides (M_r 15000–180000) are part of the complex and only eight of them are found in the RNA polymerase preparation. Only five polypeptides are always present with the same yield. They are probably the subunits of the RNA polymerase. The molecular weight of these subunits ranged from 15000–69000, even if the isolation of the enzyme was performed in the presence of protease inhibitors.