Studies of the binding of Escherichia coli RNA polymerase to DNA. V. T7 RNA chain initiation by enzyme-DNA complexes

Abstract

Initiation of T7 RNA chains by Escherichia coli RNA polymerase-T7 DNA complexes has been followed using incorporation of λ-32P-labeled ATP and GTP to determine the relation between the enzyme binding sites and RNA chain initiation sites on the T7 genome. If the period of RNA synthesis is limited to less than two minutes, the stoichiometry of RNA chain initiation can be measured in the absence of chain termination and re-initiation. About 70% of the RNA polymerase holoenzyme molecules in current enzyme preparations are able to rapidly initiate a T7 RNA chain. The ratio of ATP- to GTP-initiated T7 RNA chains is not altered by variations in the number of enzyme molecules added per DNA, nor by alterations in the ionic conditions employed for RNA synthesis. This suggests that RNA chain initiation sites are chosen randomly through binding of RNA polymerase to tight (class A) binding sites on T7 DNA. Although there are only about eight class A binding sites per T7 genome, many T7 RNA chains can be initiated per genome, hence, RNA polymerase can pass rapidly from other (class B) sites on the T7 genome to locate an RNA chain initiation site and these initiation sites are rapidly re-used. In the presence of rifampicin, the period of time allowed for RNA chain initiation can be reduced to a very short interval. At very high rifampicin concentrations and in the presence of excess RNA polymerase, T7 RNA chain initiation appears to be restricted to two ATP- and one GTP-initiated chains per genome. Since a major fraction of this T7 RNA is thought to be initiated at the early promoter region, this suggests that there are multiple binding and initiation sites in that region. © 1972.