Studies with the RNA polymerase I. Factors affecting the binding of nucleic acid polymers to the enzyme

Abstract

The association of RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) with DNA or RNA as well as some of the factors which affect the enzyme-polymer complex have been examined. 1. 1. The 13-S form of the enzyme was bound to transfer RNA (tRNA). Binding of slightly more than 1 tRNA per enzyme molecule was observed. 2. 2. An order of displacement for nucleic acid polymers was established by experiments which demonstrated the displacement of one polymer from the enzyme by a second polymer. The order was: single-stranded sonicated DNA ≧ double-stranded sonicated DNA > single-stranded DNA > tRNA > native DNA. 3. 3. Ionic strength affected the association of enzyme and polymer. Association was greatest at low ionic strength; in centrifugation experiments, (NH 4) 2SO 4 at 0.066 M caused a 90% dissociation of the enzyme-polymer complex. In kinetic experiments with varying DNA concentrations, (NH 4) 2SO 4 produced noncompetitive inhibition. 4. 4. Divalent cations decreased association. In the absence of divalent cations and in the presence of EDTA, 20–25% more RNA or DNA was bound to the enzyme than in the presence of divalent cations. 5. 5. Nucleoside triphosphates at elevated concentrations produced dissociation of the RNA-enzyme complex. The order for the displacement effect was ATP > GTP > UTP > CTP. Nucleoside triphosphates also produced either dissociation or aggregation of the DNA-enzyme complex. The order for this effect was GTP > ATP > CTP > UTP. A divalent cation was required for this nucleotide activity with both RNA and DNA. 6. 6. Nucleoside triphosphates at elevated concentrations also inactivated the enzyme. The order of activity for this effect was GTP > ATP > CTP > UTP. 7. 7. The effects of the nucleotides lead to a prediction that a site exists on the RNA polymerase which interacts preferentially with the purine nucleotides. It is suggested that this may be the binding site for the 5′-terminal nucleoside triphosphate.