The activity of the sigma subunit of the RNA polymerase of Bacillus subtilis decreases markedly during the first 2 hr of sporulation. Moreover, sigma activity remains deficient throughout the Early during bacterial sporulation, RNA polymerase of Bacillus subtilis undergoes a change in template specificity.RNA polymerase purified from vegetative cells transcribes both phage ye DNA and poly(dA-dT) in vitro, while enzyme from sporulating B. subtilis actively transcribes only the synthetic template (1). Vegetative RNA polymerase holoenzyme consists of f' and # subunits of about 150,000 daltons, two a subunits of 42,000 daltons, and a sigma subunit of 55,000 daltons (2). Phosphocellulose chromatography dissociates the sigma factor, a subunit necessary for transcription of ye DNA, from the core of polymerase (#'fla2) that actively transcribes poly(dA-dT). On the other hand, RNA polymerase core enzyme purified from cells harvested during the sixth hour of sporulation is missing a # subunit and contains a polypeptide of 110,000 daltons (2). Moreover, this enzyme from sporulating cells neither binds vegetative sigma factor (3) nor responds to sigma in vitro (2). This finding suggested a possible mechanism for the change in template specificity of polymerase. Alteration of the core of RNA polymerase would prevent functioning of the sigma factor and thereby cause the loss of vegetative template specificity early during sporulation.To test this idea, we purified RNA polymerase at various stages of growth and sporulation. We report that the change in template specificity occurs during the first hour after the end 1865 of logarithmic growth, but that alteration of the core of polymerase does not commence until the second hour of sporulation. Brevet (in preparation) has independently made the same observation. Thus, the known alteration of core enzyme occurs too late to account for the loss of sigma activity. Furthermore, we find, by purifying RNA polymerase from a mixture of vegetative and sporulating bacteria separately labeled with two different radioisotopes, that the alteration of the # subunit may be accounted for by proteolytic cleavage in vitro during purification and that sporulating cells and spores contain polymerase with # subunits of molecular weight identical to that of vegetative polymerase. Possible mechanisms for the loss of sigma factor activity early during sporulation are discussed. METHODSMedia and Sporulation. Wild-type B. 8ubtilis strain NCTC 3610 (ATCC 6051), a Marburg strain, was used for ill the experiments. Vegetative and sporulating cells were grown in 121 B medium by the method of Sonenshein and Roscoe (4). For radioactive labeling of cells, the radioactive precursor was added during early logarithmic growth.The end of logarithmic growth is the start of the sporulation process and is defined as To. Refractile prespores began to appear after 5-6 hr, i.e., T5 and T6, and dormant spores were released at about T10-TI2. The total extent of sporulation after 24 hr was about 95%.Buffers were der...
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...
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