In Vitro Synthesis of Ribosomal RNA by Bacillus subtilis RNA Polymerase

Abstract

The ability of RNA polymerase holoenzyme to synthesize rRNA in vitro is not lost after extensive purification. RNA polymerase core enzyme, however, which is missing the a, factor, synthesizes little rRNA in vitro.RNA polymerase purified from wild-type sporulating cells synthesizes little rRNA in vitro, while the in vitro synthesis of rRNA by RNA polymerase from stationary phase cells of the sporulation-defective mutant fr .10 is apparently unimpaired.While the ribosomal RNA (rRNA) genes of Bacillus subtilis are actively transcribed during logarithmic growth, the synthesis of rRNA is abruptly turned off early during the process of sporulation (1). This turn off is prevented, however, in a mutant known as rfr 10, which is resistant to the drug rifampicin (1, 2). Rfr 10 cells sporulate with less than 5% the frequency of wild-type cells (2). To test the idea that the turn off of rRNA genes is due to the alteration of RNA polymerase during spore formation (2-5), we have looked for the synthesis of rRNA in vitro. Since rRNA accotints for 15-45% of the total RNA synthesized at any given time in rapidly growing bacteria (6-9), it seemed likely that rRNA would comprise a substantial fraction of RNA transcribed in vitro from B. subtilis DNA. We report here that highly purified RNA polymerase from vegetative cells of B. subtilis initiates the synthesis of rRNA in vitro. The rRNA is copied from the heavy (H) strand of B. subtilis DNA and accounts for at least 8% of the RNA synthesized in vitro. We also report experiments on the in vitro synthesis of rRNA by RNA polymerase that is purified from wild-type sporulating cells and from stationary phase cells of the oligosporogenous mutant rfr 10. Haseltine (manuscript in preparation) has independently discovered that purified Escherichia coli RNA polymerase also synthesizes rRNA in vitro. In Vitro Synthesis of RNA. The reaqtiori mixtures contained 0.04 M Tris -Cl (pH 7.9); 0.01 M MgCl2; 1 mM EDTA; 1 mM dithiothreitol; 0.5 nig/ml bovine serum albumin; 0.15 mAM ATP, 0.15 mM CTP, 0.15 mM GTP, and 0.05 mM[8H]UTP (specific activity as indicated in the legends); 0.4 mM potassium phosphate; 16 ug/ml of B. subtilis DNA; indicated amounts of RNA polymerase. After incubation at 37°C for 15 min, RNA was extracted with phenol and precipitated with ethanol as described (13).Hybridizations in Liquid. Hybridization reactions were performed at 69°C for 5 hr with H-and L-strand DNA and 3 hr with alkali-denatured DNA. After hybridization, the reaction mixtures were incubated with 0.8 ml of heat-treated RNase A (Sigma, 5 X crystallized) in 2 X SSC (0.30 M NaCl-0.030 M sodium citrate) (10 ag/ml) for 30 min at 34°C. Hybrids were collected on Schleicher and Schuell 13-6 filters and washed with 60 ml of buffer containing 0.01 M Tris HCO (pH 7.5) and 0.5 M KCl. After drying, the radioactivity retained on the filters was measured in a liquid scintillation counter. For hybrids containing radioactivity from both 'H and 32p, correction was made for the 1% crossover of 32p into the "H channel. RESULT...

An RNA Polymerase Mutation Causing Temperature-Sensitive Sporulation in Bacillus subtilis

“…However, the initial derangement in postexponential pulse-labeled profiles of RNA are similar in both mutants. RNA (17,18). It has been demonstrated that a rifampin-resistant mutation of RNA polymerase (rfr-10) that blocks sporulation at stage 0 causes continued transcription of 4e DNA and ribosomal genes (8,17,18) during postexponential time periods.…”

“…RNA (17,18). It has been demonstrated that a rifampin-resistant mutation of RNA polymerase (rfr-10) that blocks sporulation at stage 0 causes continued transcription of 4e DNA and ribosomal genes (8,17,18) during postexponential time periods. Wild-type sporulating cells do not transcribe Oe DNA or ribosomal genes in vivo, and RNA polymerase prepared from these cells exhibits similar specificity in vitro (8,17,18).…”

Isolation of a New RNA Polymerase-Binding Protein from Sporulating Bacillus subtilis

“…All steps were at 0-4o unless otherwise noted. Each gram of radioactively labeled cells was mixed with 5 ml of buffer G, 6 ml of 120-.um glass beads, 0.25 ml of phenylmethyl sulfonyl fluoride solution (6 mg/ml of 95% ethanol), and 1 drop tri-nbutylcitrate, and disrupted 3-4 min in a Braun shaker. After the liquid was decanted, the beads were washed on a filter funnel with several ml of buffer G; the filtrate and decanted liquid were combined and centrifuged at 110,000 X g for 1.5 hr.…”

“…3) and the synthesis of ribosomal RNA in vitro (6). Attempts to demonstrate a role for the 70,000-dalton binding protein in the transcription of sporulation genes have not yet been successful.…”

Template specificity of transcription during sporulation of Bacillus subtilis

“…1). Particular emphasis [2][3][4][5][6][7] 8 has been put on transcription and, more recently, translation , trying to find a biochemical basis for the dramatic alterations in the pattern of gene expression which lead.to spore formation.…”