Inhibition of the RNA polymerase-catalyzed synthesis of RNA by daunomycin. Effect of the inhibitor on the late steps of RNA chain initiation.

In vitro RNA synthesis from the A promoters of T7 bacteriophage by Escherichia coli RNA polymerase is strongly inhibited by daunomycin. Under the conditions of the assay for total RNA synthesis, daunomycin has no effect on the formation of the binary enzyme-template complex. The major inhibitory effect may be exerted during incorporation of the first few nucleotides into the nascent RNA chain. We report here that daunomycin has little effect on the substeps leading to the formation of abortive dinucleotide from the A promoters. A large part of the inhibitory effect of daunomycin is therefore specifically targeted toward a very small final portion of the process of initiation, which may be described as the addition of nucleotides to the initial dinucleotide (and its subsequent translocation) until a stable ternary complex has been formed. The level at which this inhibition is exerted is more precisely defined by examining the effect of daunomycin on the synthesis of the first few oligonucleotides synthesized from the A3 promoter. The major inhibitory effect of daunomycin is found to be specifically exerted during the transformation of the initial dinucleotide to the corresponding trinucleotide. The remainder of the inhibition may be evenly divided among a large number of nucleotide addition steps that transform the nascent trinucleotide to a completed RNA chain. One of the fundamental levels at which transcription is controlled is initiation. In slow-start promoters, e.g. the lac UV5 promoter, the late stages of initiation (when the enzyme has the option of either producing an abortive di- or trinucleotide or proceeding with the formation of a stable ternary complex) may be involved in this control. Inhibitors which specifically act on this stage of initiation may thus prove useful for the study of such systems, as well as transcription in general.