Mechanism of RNA polymerase action: Characterization of the DNA-dependent synthesis of polyadenylic acid

Abstract

Purified preparations of DNA-dependent RNA polymerase from Escherichia coli catalyse a DNA-dependent synthesis of long-chain polyadenylic acid when ATP is the only ribonucleoside triphosphate added to the reaction. Polyadenylic acid synthesis differs from DNA-dependent synthesis of complementary RNA in three important respects. First, the chain-length of the polyadenylic acid product is greater by 5 to 10 times than that of the deoxythymidylic acid sequence in DNA which serves as template. Secondly, addition of one, two or all three of the other ribonucleoside triphosphates to the reaction at low concentrations (less than 2×10 −6 M ) brings about a striking inhibition of polyadenylic acid synthesis. In the presence of all four of the ribonucleoside triphosphates, no polyadenylic acid is produced. Inhibition by any given ribonucleoside triphosphate is dependent on the presence in the DNA template of the nucleotide base complementary to that inhibiting triphosphate. Finally, only single-stranded DNA serves to direct polyadenylic acid synthesis. It is proposed that DNA-dependent polyadenylic acid synthesis is due to a reiterative copying of short sequences of thymidylic acid residues in the DNA template rather than to a separate polyadenylic acid polymerase. The inhibition of this synthesis by the other ribonucleoside triphosphates may be due to fixation of the enzyme on the template at a deoxynucleotide base complementary to the base of the inhibiting triphosphate, thus preventing further copying of thymidylic acid sequences. The relevance of these findings to the general mechanism of polynucleotide synthesis by this enzyme is discussed.