SUBSTRATE REQUIREMENTS FOR INITIATION AND CHAIN GROWTH OF ENZYMICALLY SYNTHESIZED RNA

RNA synthesis was followed in reaction mixtures containing Escherichia coli RNA polymerase, T4 DNA as template and the four ribonucleoside 5′-triphosphates as substrates in varying concentrations. All RNA products longer than pentanucleotides were recovered. The rate of RNA chain growth as well as the number of active polymerase molecules were found to increase with substrate concentration. The substrate requirements for RNA chain initiation are different for three classes of polymerase molecules. (1) About 20% of the polymerase molecules synthesizing 85% of the product initiate RNA synthesis during the first minute of reaction and then function for about 25 minutes; they require up to 20 mμmoles of substrates/ ml. to become active. (2) About 40% of the polymerase molecules initiate and terminate RNA synthesis during the first minute of reaction, and require up to 400 mμmoles of substrates/ml. to become active; this substrate requirement is lower if polymerase and DNA are incubated before the addition of substrates. (3) About 40% of the polymerase molecules also function for less than one minute but initiate RNA molecules throughout the first 20 minutes of reaction and require less than 20 mμmoles of substrates/ml. for activation; they cannot be induced to start earlier by pre-incubation with DNA in absence of substrates. The functional heterogeneity of the polymerase observed here is compared with the physical heterogeneity of polymerase-DNA complexes described by other authors. The comparison suggests that the polymerase molecules catalyzing synthesis of the short RNA chains form dissociable complexes with the DNA in absence of substrates. © 1969.