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Ribonucleic Acid Polymerase from Micrococcus luteus

III. THE EFFECT OF SUBSTRATE, METAL ION, AND TEMPERATURE ON THE HOMOPOLYMER-DIRECTED REACTION

From the ¹ From the Department of Radiological Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205

Factors influencing synthetic rates by highly purified RNA polymerase from Micrococcus luteus with single and double stranded homopolymer templates have been investigated. The results indicate different reaction mechanisms depending on the number of strands of the template. Double stranded homopolymer templates show typical Michaelis-Menten kinetics with respect to the nucleoside triphosphate concentration whereas single stranded homopolynucleotides show sigmoidal kinetics. Double stranded deoxyribosyl templates are also affected less by lower temperatures than single stranded deoxyribosyl templates, suggesting that "melting" of the DNA template is not rate-limiting in this system. The reactions directed by single and double stranded templates also differ in their response to manganese concentration. For AMP incorporation, single stranded templates show a sharp manganese optimum whereas double stranded templates show a broad optimum. For uridine monophosphate incorporation, both single and double stranded templates show a sharp optimum. These results indicate functions for manganese other than in the formation of a nucleoside triphosphate-manganese complex. It is suggested that the results reflect a rate-limiting step involving product release (i.e. translation of the product from the active site of the enzyme) and that release is inhibited by high manganese concentrations.

The sigmoidal dependence of the reactions directed by single stranded templates upon substrate concentration indicates a concentration-dependent interaction between the substrate and some other component of the reaction. This component is not manganese since maintaining a stoichiometric ratio between Mn⁺⁺ and ATP in the r(U)_n-directed reaction does not completely abolish the shape of the curve. Further, several lines of evidence suggest that the stacking interaction between the nucleoside triphosphate substrate and the template also does not account for the shape of the curve. Only the presence of a second template strand eliminates the sigmoidal profile of the substrate curves.

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