The molecular mechanism of the enzymic reaction in RNA synthesis

Abstract

1. 1. The separation of ribonuclease from DNA-dependent RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was carried out by fractionation at narrower ranges of ammonium sulfate concentration than in the original method. 2. 2. The polymerase preparation thus obtained exhibited two different sedimenting behaviors in sucrose gradient centrifugation. 3. 3. The RNA synthesized in the enzymic reaction exhibited a heterogeneous distribution in molecular sizes: 8 S, 12 S, 18 S and 26 S. At early stages in the reaction the largest product was dominant at least up to 15 min. The rate of polynucleotide chain growth was calculated to be roughly 13–14 nucleotide monomers/sec. 4. 4. Persistence of the reaction was determined by the relative ratio of enzyme to primer DNA. Successive additions of enzyme made the reaction proceed linearly. 5. 5. The RNA polymerase-DNA complex was fractionated by zone electrophoresis and was proved to be essential for the reaction. 6. 6. The molecular hypothesis on the primer-dependent nature of the reaction was theoretically considered, on the basis of the conclusion that RNA polymerase could bind specifically to DNA prior to the reaction and that only the active complex participated in the process.