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Inhibition of Deoxyribonucleic Acid-directed Ribonucleic Acid Polymerase by Extracts of Steroid-induced and Noninduced Pseudomonas testosteroni

From the ¹ From the Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The synthesis of ribonucleic acid, catalyzed by highly purified preparations of Escherichia coli RNA polymerase under the direction of native calf thymus DNA, was powerfully inhibited by high molecular weight fractions obtained from extracts of steroid-induced and noninduced Pseudomonas testosteroni by gel filtration on Sephadex G-100. When heat-treated DNA was used as primer, the inhibitory effect of these macromolecular fractions was no longer observed. Likewise, polyadenylate formation catalyzed by the polymerase was not affected by the macromolecular fractions with either native or heat-denatured DNA as a primer.

The major components of the macromolecular fractions are DNA, RNA, and protein. Analysis of the mechanism of this inhibitory effect on RNA synthesis revealed that it was probably not the result of the breakdown of input nucleoside triphosphates or the breakdown of the product RNA. The inhibitory activity of the macromolecular fractions was greatly enhanced upon heating. The inhibition was observed when the system was saturated with primer DNA, and also when the inhibitor was added after exposure of the enzyme to primer DNA.

Contrary to the reports of others, our study showed that the inhibitory activity of the macromolecular fraction derived from noninduced cells was not reversed by prior incubation with testosterone. Furthermore, no quantitative difference was observed between the inhibitory potency of macromolecular fractions derived from steroid-induced and noninduced cells. These results do not support the claim that experiments of this type permit direct demonstration of changes by enzyme inducers of the priming capacity of DNA for RNA synthesis.

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