DNA-dependent synthesis of RNA by Escherichia coli RNA polymerase: release and reinitiation of RNA chains from DNA templates

Abstract

RNA synthesis in an in vitro RNA polymerase system at low ionic strength soon ceases, due to inhibition by accumulated RNA. Measurement of RNA chain initiation by the incorporation of gamma-(32)P-ATP and GTP with native T2 or T4 DNA as template shows that only one RNA chain is formed per molecule of enzyme added. In contrast, when the polymerase reaction is carried out in 10 mM Mg(++) and 0.2 M KCl, RNA synthesis proceeds nearly linearly for hours, resulting in a marked increase in accumulated RNA. Incorporation of gamma-(32)P-ATP also proceeds throughout the course of the reaction and the number of chains initiated per molecule of enzyme is increased severalfold. Most RNA chains formed are released from the DNA template as free RNA. Polymerase is released also in this process and acting catalytically reinitiates new chains. Rifampicin inhibits initiation of RNA synthesis and also blocks reinitiation of RNA chains without affecting growth of RNA chains already initiated.