The Role of Deoxyribonucleic Acid in Ribonucleic Acid Synthesis: XIV. A STUDY OF THE INITIATION OF RIBONUCLEIC ACID SYNTHESIS

Abstract

Abstract Initiation and growth of RNA chains in the DNA-dependent RNA polymerase reaction have been studied by measuring the incorporation of γ-32P-labeled nucleoside triphosphates and 14C- (or α-32P) nucleoside triphosphates into RNA. The ratio of total nucleotide of RNA synthesized to total initiation is a measure of the average chain length of the RNA produced in the polymerase reaction. The length of RNA chains synthesized is independent of nucleoside triphosphate concentration and of enzyme concentration after extensive RNA synthesis, since both initiation and synthesis increase proportionally. However, the average RNA chain length is dependent on DNA template concentration. The rate of RNA synthesis increases more rapidly than RNA chain initiation as the DNA concentration is elevated. The specificity of initiation of chains with either ATP or GTP is independent of the above three parameters. The nucleotide penultimate to the nucleoside triphosphate end has been determined with Escherichia coli and T4 DNA as templates. All four nucleotides are found in this position in a nonrandom distribution, although there is a preponderance of pyrimidine over purine nucleotides in this second position of a growing RNA chain. Of a number of inhibitors that have been examined for their differential effects on initiation and chain growth, actinomycin D hardly affects initiation of RNA chains but markedly inhibits the synthesis of RNA, thus leading to a marked decrease in the average chain length of the RNA products. With denatured DNA as template the average chain length is markedly decreased, since the number of initiations increases as much as 10-fold and synthesis is slower. With either denatured DNA or tobacco mosaic virus RNA as primer, initiation with GTP predominates over that with ATP. From the known molecular weight of RNA polymerase, it can be calculated that approximately 1 RNA chain is formed by 1 molecule of enzyme. With denatured DNA, reinitiation can occur such that several chains are formed per molecule of enzyme.