The Role of Deoxyribonucleic Acid in Ribonucleic Acid Synthesis: XVII. MULTIPLE ACTIVE SITES OF ESCHERICHIA COLI RIBONUCLEIC ACID POLYMERASE

Abstract

Abstract The over-all reaction catalyzed by RNA polymerase has been studied in discrete steps. These steps include (a) the binding of polymerase to DNA, (b) the binding of ribonucleoside triphosphates to RNA polymerase, (c) the initiation of RNA chains measured by 32PPi exchange reaction, and (d) the elongation of RNA chains as measured by nucleotides incorporation. A number of chemical reagents have been used to inactivate one or more of these four reactions selectively. In the presence of reagents which react with sulfhydryl groups, all the reactions except the binding of ribonucleoside triphosphates to the enzyme are quantitatively inactivated. Treatment of the enzyme with β-naphthoquinone-4-sulfonic acid and chlorofluoro-p-benzoquinone results in preparations unable to bind ribonucleoside triphosphates and thus unable to initiate or elongate RNA chains. The ability of the enzyme to bind to DNA is unaffected by this treatment. In contrast to these reagents, diazo-1H-tetrazole and rose bengal (plus photooxidation) treatment of RNA polymerase results in enzyme preparations capable of binding DNA, binding ribonucleoside triphosphates, and catalyzing a rapid DNA-dependent 32PPi exchange reaction. However, such enzyme preparations are unable to elongate RNA chains. The properties of the chemically altered enzyme have been examined in the analytical ultracentrifuge. No marked alteration in the sedimentation values were obtained with reagents such as β-naphthoquinone-4-sulfonic acid or diazo-1H-tetrazole. However, in the presence of p-chloromercuribenzoate, a small subunit of 2 S was dissociated from the enzyme. A study of the binding reaction between RNA polymerase and nucleoside triphosphates has been carried out. RNA polymerase of Escherichia coli will bind between 8 and 9 moles of nucleoside triphosphates per mole of enzyme (molecular weight, 340,000) and this reaction is stimulated by the addition of Mg2+ or Mn2+. In the absence of DNA the enzyme interacts identically with ATP, GTP, CTP, and UTP. The binding of 14C-ATP was displaced by an excess of unlabeled GTP or CTP, and the binding of 14C-GTP was completely displaced by an excess of unlabeled ATP or CTP. In the presence of DNA, the binding reaction between enzyme and nucleoside triphosphate is more restrictive. In the presence of thymus DNA, GTP and ATP are bound more preferentially by the enzyme while in the presence of the copolymer of deoxyadenylate and deoxythymidylate ATP is bound preferentially.