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.

A kinetic study on the mechanism of inhibition of RNA synthesis catalyzed by DNA-dependent RNA polymerase. Differences in inhibition by ethidium bromide, 3,8-diamino-6-ethylphenanthridinium bromide and actinomycin d.

The mechanism of inhibition of RNA polymerase-catalyzed synthesis of RNA by actinomycin D and the phenan-thridinium derivatives ethidium bromide and 3,8-diamino-6-ethylphenanthridinium bromide (DEMB) is examined. A general kinetic equation describing the dependence of RNA synthesis on DNA template concentration is derived and distinct expressions corresponding to various possible mechanisms of inhibition are subsequently obtained by introducing into the equations assumptions as appropriate for the individual mechanisms. The fitting of the experimental results of inhibition into the resulting equations suggested that the ethidium bromide and DEMB inhibit RNA polymerase by forming an inhibitor-template complex which interferes with enzyme recognition of, and binding to, appropriate sites on the template (binding inhibition). The fitting of the dependence of the rate of RNA synthesis on the bound-inhibitor to DNA ratios to the derived kinetic expressions also allows a tentative distinction to be made as to whether ethidium bromide and DEMB interfere with RNA synthesis by a mechanism of 'partial' or 'complete' inhibition.

Circular dichroism study of the conformation of ultraviolet-irradiated ribonuclease A.

RNAase A irradiated by ultraviolet light at 254 nm shows a linear dependence between loss of activity and destruction of cystine. At least one of the cystine modified forms in irradiated RNAase is catalytically active. Circular dichroism spectra of irradiated RNAase show a marked decrease in ellipticity between 210 nm and 230 nm, an increased ellipticity between 230 nm and 240 nm, and a blue shift of the 210-nm minimum toward 205 nm. These circular dichroism changes indicate a pariial disorganization of the native secondary and tertiary changes with irradiation. The temperature dependency of the circular dichroism shows the irradiated enzyme to be conformationally less stable to thermal perturbation than native RNAase. Differences in the polypeptide conformations of unirradiated RNAase denatured by heat and sodium dodecylsulfate, and irradiated RNAase treated with heat and sodium dodecylsulfate are discussed.

Thermal denaturation of the DNA-ethidium complex. Redistribution of the intercalated dye during melting.

The temperature dependence of the circular dichroism of the DNA-ethidium bromide complex at elevated temperatures provides evidence that the optical activity of the complex near 307 nm originates from interactions between intercalated dye molecules while the optical activity near 515 nm results from singly intercalated ethidium bromide molecules. The behavior of the circular dichroism of the complex at elevated temperatures also explains the higher ellipticities near 307 nm which characterize complexes formed between ethidium bromide and denaturated DNA. Finally the circular dichroism data indicate that the melting of the complex takes place in a stepwise manner with some DNA regions, probably AT-rich regions, dissociating first. The implications of these findings regarding the inhibiting effect of ethidium bromide on the function of DNA polymerase are examined.