Nucleotide-Dependent Inactivation of RNA Polymerase from Bacillus brevis

Abstract

RNA polymerase has been purified from vegetative cells of Bacillus brevis and resolved into 'core' enzyme and sigma factor. The purified enzyme is rapidly inactivated by incubation at low temperatures in the presence of 1-2 mM ATP, dATP, or NAD+, while other nucleotides at this concentration have little or no effect. Inactivation is not accompanied by the incorporation of an adenylyl or phosphoryl moiety into RNA polymerase; nevertheless, it is essentially irreversible. DNA, high concentrations of glycerol, as well as low concentrations (1 mM) of orthophosphate protect RNA polymerase from the nucleotide-dependent inactivation.A similar inactivation of RNA polymerase in the presence of ATP is observed with crude preparations from Bacillus subtilis and Bacillus polymyxa. This phenomenon may represent a novel mode of regulation of transcription that does not involve a covalent modification of RNA polymerase or its interaction with other protein factors, but rather is due to a structural transition to an inactive form induced by small molecules.The transcription of specific genes by bacterial RNA polymerases is known to be modulated by protein factors that interact either with the enzyme itself or with the DNA template (1-3). Under conditions of drastic physiological change, as upon bacteriophage infection or during sporulation, a more general control of transcription is seen that often involves the covalent modification of RNA polymerase (4, 5). Between these two extreme types of regulation lies the noncovalent modification of RNA polymerase by small molecules, such as the inhibition by peptide antibiotics during the early stages of sporulation (6). In this paper, we describe a phenomenon that may also belong in this category-although its physiological significance is still obscure-namely, the inactivation of RNA polymerase from Bacillus brevis in the presence of specific nucleotides. precipitated with ammonium sulfate (70% saturation), and redissolved in Buffer A containing 50% glycerol for storage at -20°(Fraction IV). The purification procedure is summarized in Table 1. MATERIALS AND METHODS Growth ofCrude preparations of RNA polymerase were obtained from sonic extracts of B. polymyxa and B. brevis by centrifugation at 100,000 X g for 1 hr and fractionation with ammonium sulfate.Enzyme Assays. RNA polymerase was assayed at 370 for 15 min in the presence of 0.1 mM [5-3H]UTP (10 Ci/mol), 1 mM (each) of ATP, GTP, and CTP, 10 mM MgCl2, 2 mM MnCl2, 10 mM 2-mercaptoethanol, bovine serum albumin (0.5 mg/ml), and DNA (0.1 mg/ml of salmon-sperm DNA unless otherwise indicated), in a final volume of 0.2 ml. The reaction was terminated by the addition of cold trichloroacetic acid (0.3 N), and the precipitate was washed with trichloroacetic acid, then with ethanol, on discs of glass-fiber filter paper (Whatman GF/C). The radioactivity was determined in a liquid scintillation spectrometer in a toluene-based scintillation fluid. 1 Unit of RNA polymerase activity is defined as the amount of enzyme that catalyzes the inc...

Effect of GTP and Other Nucleoside Triphosphates on GMP Incorporation by Isolated Cortical Nuclei

“…Partial and similar results were reported by Novello and Stirpe (1969). Sarkar and Paulus (1972) reported an inactivation by ATP of a purified bacterial RNA po~ymerase. However, there a:e several differences between their report and our results.…”

Effect of Gramicidin S on the Transcription System of the Producer Bacillus brevis Nagano

“…We also consider important the finding that the antibiotics are found to bind B. brevis Nagano DNA with an efficiency analogous to their ability to inhibit transcription in vitro (gramicidin S > tyrocidine > tyrothricin > linear gramicidin). From the information available these results could argue for a stronger affinity of gramicidin S for B. brevis Nagano template, since it has been reported that 10 pg gramicidin S can inhibit transcription in B. brevis ATCC 8 185 (a non-producer of gramicidin S) only to about 23 %, whereas tyrothricin, tyrocidine, or linear gramicidin (produced by the B. brevis ATCC 8 185 strain) tested at the same concentration caused 96%, 100% and 90% inhibition of transcription respectively (Sarkar & Paulus, 1972a).…”

“…3 b), and the molar ratios were calculated from the densitometer tracings. The major components had approximate molecular weights of 150000 f: 10000; 65000 5000, and 38000 +_ 1000, and occurred in molar ratios of about 2.6 : 1-3 : 3 i-0.4, thus corresponding well to the p + p', CT, and a subunits of RNA polymerase from B. brevis ATCC 81 85 (Sarkar & Paulus, 19726) and B. subtilis (Avila et al, 1971). Subunit a was found in higher ratios than subunits fl p' in a number of preparations, and this was attributed to impurities running very close to the a band of the gels (Fig.…”

“…It was shown that tyrocidine could inhibit RNA synthesis in B . brevis ATCC 8185 in vivo and in vitro (Sarkar & Paulus, 1972a;Schazschneider et al, 1974;Ristow et al, 1975a). In the in vitro studies a complex was demonstrated between the antibiotic and the DNA (Schazschneider et al, 1974).…”

“…On the other hand, linear gramicidin, which could also inhibit RNA synthesis in vitro (Ristow et al, 1975b;Paulus & Sarkar, 1976), apparently by interacting specifically with the DNA-dependent RNA polymerase (Sarkar et al, 1979), could weaken the DNA-tyrocidine complex, operating antagonistically to tyrocidine (Ristow, 1977;Chakraborty et al, 1978). Since both antibiotics are produced by the same strain in B. brevis they were suggested as possible regulators of gene expression during differentiation in their producer (Sarkar & Paulus, 1972a;Ristow et al, 1979).…”