Inhibition of the RNA polymerase-catalyzed synthesis of RNA by marcellomycin. Preferential interference of the inhibitor with the stabilization of the ternary promoter-RNA polymerase-nascent RNA complex.

Marcellomycin is a strong inhibitor of the Escherichia coli RNA polymerase-catalyzed synthesis of RNA from the strong A promoters of bacteriophage T7 DNA. Marcellomycin inhibits preferentially the last phase of transcription initiation. During this phase a stabilized ternary complex is formed consisting of RNA polymerase, DNA template, and a nascent RNA oligonucleotide about 11 nucleotides long, resulting from the extension of the RNA dinucleotide component of the corresponding early ternary complex. Marcellomycin is also responsible for minor inhibition of the formation of the open binary RNA polymerase-template complex, which serves as the precursor of the ternary complex. These findings suggest that marcellomycin may be a potentially useful tool in the study of the late stages of transcription initiation. The present findings may also contribute to a better overall understanding of the mode of drug action at the level of individual genes.

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.

On the inhibition of the RNA polymerase-catalysed synthesis of RNA by daunomycin. Interference of the inhibitor with elongation and pre-longation steps.

The inhibition of the RNA polymerase-catalyzed synthesis of RNA by daunomycin was examined. Saturation binding of daunomycin to the template leads, as expected, to complete inhibition of RNA synthesis as a result of daunomycin interference with enzyme-template interactions. However at concentrations of the inhibitor below saturation formation of the enzyme-template complex remains remarkably undisturbed, while both the transformation of this complex to an elongating complex and the elongation of the nacsent RNA chains are substantially inhibited. Clearly, daunomycin interferes with a number of different substeps of RNA synthesis and inhibits the synthesis by different mechanisms depending on the amount of inhibitor bound to the template. Elucidation of the mechanism of inhibition at low daunomycin concentrations may be a prerequisite for a better understanding of the mechanism of the pharmacological action of the drug.