Role of RNA Polymerase in Sporulation

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...

Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Nucleotide-Dependent Inactivation of RNA Polymerase from Bacillus brevis

Sarkar¹,

Paulus²

1972

“…The availability of in vitro systems for the study of transcription has been of prime importance in elucidating mechanisms of gene regulation in prokaryotes (1,2). The lack of similar eukaryotic systems has left many important problems unresolved.…”

mentioning

confidence: 99%

DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.

Manley¹,

Fire²,

Cano³

et al. 1980

1,159

656

We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The (4,5). Recently, Weil et al. (6) demonstrated that accurate initiation of transcription occurs on exogenously added DNA at a known viral transcription start site when purified RNA polymerase II is mixed with a cytoplasmic extract. Here we describe a simple system consisting primarily of a soluble whole-cell extract that accurately initiates transcription on exogenously added DNA templates. We feel that this system will prove useful for studying the biochemical mechanisms involved in the control of transcription and gene expression in mammalian cells.MATERIALS AND METHODS Preparation of DNA. Adenovirus serotype 2 (Ad2) virus and DNA were prepared as described (7). Restriction endonuclease DNA fragments Bal I-D [21.5-28.3 map units (m.u.)] and Bal I-E (14.7-21.5 m.u.) were inserted into plasmid pBR322 with BamHI linkers and cloned (8). Sma I-F fragment (2.9-11.3 m.u.) was similarly inserted into pBR322 but with EcoRI linkers (gift of Kathleen Berkner and Frank Laski). Plasmid DNA was prepared by standard protocols (9).Preparation of HeLa Cell Extracts. Cell lysates were prepared according to the method of Sugden and Keller (10 decanted so as not to disturb the pellet and precipitated by addition of solid (NH4)2SO4 (0.33 g/ml of suspension). After the (NH4)2SO4 was dissolved, 0.01 ml of 1 M NaOH per 10 g of (NH4)2SO4 was added, and the suspension was stirred for an additional 30 min. The precipitate was collected by centrifugation at 15,000 X g for 20 min and resuspended with 'Ao the volume of the high-speed supernatant into a buffer containing 50mM Tris-HCI, pH 7.9,6mM MgCl2, 40 mM (NH4)2SO4, 0.2 mM EDTA, 1 mM dithiothreitol, and 15% glycerol. This suspension was dialyzed for 4-8 hrs against each of two changes of 100 vol of the same buffer. The dialysate was centrifuged at 10,000 X g for 10 min and the supernatant was quick frozen in small samples in liquid N2. Samples stored at -80°C retained full activity for at least 2 months. In some later experiments (e.g., the one shown in Fig. 5) the dialysis buffer was 20 mM Hepes, pH 7.9/100 mM KC1/12.5 mM MgCl2/0.1 mM EDTA/2 mM dithiothreitol/17% glycerol.In Vitro Incubations 3855

“…Thus, it is likely that sigma is essential for the viability of E. coli. Mutations (28). The mapping of the structural gene for sigma will facilitate the isolation of sigma mutants through use of localized mutagenesis (29,30).…”

Section: Resultsmentioning

confidence: 99%

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

Harris¹,

Martinez²,

Calendar³

1977

The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toWC and dnaG, at 66 min. The properties of homologous components of the transcription and translation apparatuses often vary between strains of enteric bacteria. Such natural differences provide a convenient tool for genetic mapping of these components when mutants are not available. In order to make use of such differences, investigators individually transfer small regions of the chromosome from one bacterial strain to another strain. The recipients are then screened for the desired phenotype, by using an appropriate assay. With this technique, it has been possible to map some of the genes coding for ribosomal proteins (1) as well as the gene coding for transcription termination factor rho (2). Until now, however, no genetic mapping has been reported for the RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) sigma subunit, which determines the selectivity of transcription in vitro (3). We have observed electrophoretic differences between the RNA polymerase sigma subunits of Escherichia coli strain K-12 (K), E. coli strain C, and Salmonella typhimurium, and have used these differences to map an E. coli K gene which causes synthesis of the sigma subunit characteristic of E. coli strain K.MATERIALS AND METHODS Chemicals and Buffers. Buffers and DNA-cellulose were prepared as described by Burgess and Jendrisak (4 Bacterial Strains. The bacterial strains used are described in Table 1. New merodiploid strains were constructed by transferring F' plasmids from E. coli K merodiploid strains into various Salmonella typhimurium and E. coli C strains, each containing nutritional mutations such that the presence of the F' plasmid would allow growth on minimal glucose medium. Transfer was accomplished by either of two methods. Method 1: a minimal glucose plate was spread with 0.1 ml (107-108 colony-forming units) of an overnight culture of the donor strain and 0.05 ml of the recipient strain, followed by incubation at 370 for 48 hr (13). Method 2: 0.5 ml samples of each overnight culture were inoculated into flasks containing 10 ml of minimal glucose medium supplemented with the nutritional requirements of each strain. These cultures were shaken at 370 until the optical density of the suspension was doubled. One milliliter of the recipient strain, 2 ml of the donor strain and 3 ml of LB broth were then added to a 250 ml Erlenmeyer flask and shaken very slowly at 370 for 60-90 min. The mating mixtures were centrifuged, concentrated to 10% of the initial volume, and 0.2 ml aliquots were spread on minimal glucose plates, followed by incubation at 370 for 48 hr. C...