The Enzymatic Synthesis of Ribonucleic Acid. v. the Interaction of Ribonucleic Acid Polymerase with Nucleic Acids.

Numerous reports have been published on the properties and reaction mechanism of the highly purified enzyme preparations that catalyze the deoxyribonucleic acid-directed synthesis of ribonucleic acid (l-6). The various RNA polymerases tudied, although differing in some respects, show a remarkable similarity in their cofactor requirements, reaction kinetics, physical properties, and facility to utilize both DNA (native and heat-denatured) and RNA templates in the direction of ribonucleic acid synthesis. This ability to transcribe both types of nucleic acids suggests that riboand deoxypolynucleotides combine with the same active site or sites on the enzyme. Studies in this and other laboratories have shown that RNAs are effective inhibitors of the DNA-directed reaction, and vice versa (1, 2, 7-9). In the course of our work on the inhibition of RNA synthesis in vitro by various nucleic acids, we found that the data indicated that some inhibitions were of the competitive type while others were not strictly so. Although the inhibition phenomenon was generally reproducible, certain inconsistencies were encountered which appeared to be dependent upon the order in which the nucleic acid inhibitor was introduced into the reaction system. In view of the possible role played by cellular RNAs in controlling RNA synthesis in Go (9, lo), it seemed important to examine more closely the conditions in vitro for studying the nucleic acid inhibition of RNA synthesis with the polymerase enzyme. In this report, we shall describe experiments which show that marked differences in the inhibition of RNA polymerase activity may be observed by varying the order of addition of cofactor and RNA inhibitor to the enzyme mixture, as well as by altering the reaction composition. Gros et al. (8) have reported on similar findings with Escher-f&a coli RNA polymerase. Evidence in support of enzyme-nucleic acid complex formation and its reversal will also be presented. A preliminary account of some of this work has been published (11, 12).