Studies with the Ribonucleic Acid Polymerase. II. Kinetic Aspects of Initiation and Polymerization

Kinetic studies of ribonucleic acid synthesis with the deoxyribonucleic acid dependent ribonucleic acid polymerase of Escherichia coli are presented in this paper. A nonlinear double-reciprocal plot of nucleotide incorporation as a function of the concentration of the four nucleoside triphosphates was observed. The plot became linear and the amount ot incorporation was increased, particularly at low nucleotide concentrations, if a single nucleotide was fixed at a high concentration and if this nucleotide was one of those present in a significant percentage of the 5′- terminal nucleoside triphosphate positions. This effect on the double-reciprocal plot was demonstrated with adenosine triphosphate and calf thymus deoxyribonucleic acid, guanosine triphosphate and Micrococcus lysodeikticus deoxyribonucleic acid, and adenosine triphosphate and dAT copolymer. Linear plots were also obtained if the deoxyribonucleic acid-enzyme complex was preincubated with unlabeled nucleotides to initiate ribonucleic acid synthesis and the complexes were then filtered on nitrocellulose membranes and incubated with varying concentrations of the four nucleotides. This was observed with T4, calf thymus, and M. lysodeikticus deoxyribonucleic acid. Apparent K_mvalues were obtained when three nucleotides were fixed at 0.4 mM and the fourth varied. With M. lysodeikticus deoxyribonucleic acid, where more than 85% of the 5′-terminal nucleotide is guanosine triphosphate, an apparent K_mfor guanosine triphosphate was 0.15 mM while the apparent K_mfor adenosine triphosphate, cytidine triphosphate, or uridine triphosphate was approximately 0.015 mM. With prior initiation with the four unlabeled nucleotides and M. lysodeikticus deoxyribonucleic acid, the apparent K_mfor guanosine triphosphate decreased to 0.027 mM. A tentative model has been proposed. Initiation of ribonucleic acid synthesis involves the formation of the first phosphodiester bond. The apparent K_mfor the 5′-terminal nucleotide is approximately 0.15 mM. Polymerization involves migration of the enzyme on the deoxyribonucleic acid and the addition of subsequent nucleotides. An apparent K_mfor nucleotides for polymerization is approximately 0.015 mM or one-tenth the concentration for initiation.