Enzyme der Polynucleotid-Synthese in pflanzlichen Organismen

Abstract

The isolation and purification of RNA polymerase from the blue-green alga Anacystis nidulans is described. The method consists in an initial fractional centrifugation which yields 60 — 70% of the enzyme associated with a DNA-rich fraction. It is subjected to column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) to obtain a 110 —120 fold increase in specific activity over the crude extract. The preparation is free from polynucleotide phosphorylase, kinases, phosphatases and nucleic acids. The enzyme requires DNA, a primer, as well as Mg2⊕, Mn2⊕ and the 4 ribonucleoside triphosphates for the net synthesis of polyribonucleotides. Salmon sperm DNA and native DNA from Chlorella pyrenoidosa can replace Anacystis DNA as primer. The properties of the product, however, depend to a certain extent on the primer DNA used as revealed by column chromatography of the former on methylated serum albumin. After isolation by this method, the synthesized polyribonucleotides were found to be partially stable against the action of RNase. This finding suggests a possible association of the newly synthesized RNA with the primer DNA in form of a stable complex which may function as an intermediate of the DNA directed RNA synthesis.