Ribonucleic acid synthesizing activity of spinach chloroplasts and nuclei

Abstract

Purified chloroplast and nuclei preparations from spinach leaves possess RNA polymerase activity, as measured by the incorporation of 3H-ATP into an acid-insoluble product. In both types of organelle the polymerase reaction is dependent on the presence of all four nucleoside triphosphates, Mg ++, and an ATP-generating system. The reaction is inhibited by RNase, DNase, and actinomycin D. No stimulation was found with spermidine, with ammonium sulfate, or with the plant growth regulators kinetin, indoleacetic acid, and gibberellic acid. RNA polymerase activity was higher in leaf extracts from young plants. The product of the in vitro RNA polymerase reaction was isolated by phenol extraction and sucrose density gradient centrifugation. The chloroplast product had a major peak at about 11S, and was somewhat polydisperse in the range 11–23S. The product of the in vitro nuclear reaction had a maximum at about 8S and was less polydisperse than the chloroplast product. Short-term incorporation of 3H-uridine by intact spinach plants revealed the presence of a rapidly labelled RNA fraction in the chloroplasts with sedimentation properties similar to the product of the in vitro chloroplast polymerase reaction. The corresponding rapidly labelled RNA fraction in the nuclei resembled the in vitro nuclear product but was more polydisperse in the high molecular weight regions of the gradient. In long-term, in vivo incorporation experiments the labelled RNA pattern of both chloroplasts and nuclei corresponded closely to the ribosomal and transfer RNA fractions.