Heterogeneous initiation and termination of enzymically synthesized ribonucleic acid

Abstract

The number, length, kinetics of initiation and bases in termini of RNA molecules synthesized in vitro with Escherichia coli RNA polymerase on T4 DNA templates have been determined. The RNA products were isolated by acid precipitation or by adsorption to DEAE-cellulose; this latter method allows recovery of all RNA molecules longer than pentanucleotides. The observed length distribution of RNA molecules is shown, by mathematical analysis, to be heterogeneous and non-random. It is further shown that the heterogeneity in chain lengths is due to heterogeneous RNA chain termination, which occurs with different probabilities per unit time for different classes of RNA polymerase-DNA complexes; it is not caused by strand breaks or other signals in the DNA. Three classes of RNA products have been distinguished: (1) long (acid-precipitable) RNA molecules which are initiated within the first two minutes of the reaction (preferentially with ATP) and terminated with a probability of 0.04/minute; (2) short (non-precipitable) RNA molecules which are initiated during the first 20 minutes of the reaction (preferentially with GTP) and terminated with a probability of 1.1/minute; (3) partly precipitable RNA molecules, initiated throughout the reaction with all four nucleoside-5′-monophosphates. These latter RNA products may arise when polymerase molecules skip formation of an internucleotide bond (after releasing the pyrophosphate), thus terminating and re-initiating at the same time. A model describing quantitatively the kinetics of RNA chain termination is presented in the Appendix.