Promoter recognition by phage SP01-modified RNA polymerase.

Self Cite

mentioning

confidence: 99%

“…Bacillus subtilis phage SPOl expresses its genes in a well-defined temporal sequence that appears to be controlled at the level of promoter recognition (1). Unmodified bacterial RNA polymerase selectively recognizes and binds to promoters for phage "early" genes (1-4).…”

mentioning

confidence: 99%

Distinctive nucleotide sequences of promoters recognized by RNA polymerase containing a phage-coded "sigma-like" protein.

Talkington¹,

Pero²

1979

Self Cite

“…A dramatic example is the reprogramming of promoter specificity of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) after infection with phages such as SP01 or SP82 in which a viral protein replaces the normal B. subtilis a' subunit (1)(2)(3). This type of regulatory mechanism has long been considered an attractive possibility in normal cells (4); however, although many "altered" forms of RNA polymerase have been reported (refs.…”

mentioning

confidence: 99%

Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.

Wiggs¹,

Gilman²,

Chamberlin³

1981

Preparations ofBacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B. subtilis RNA polymerase holoenzyme H) that shows a unique promoter specificity with T7 bacteriophage DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I) A large variety of molecular mechanisms are known by which organisms regulate transcription during growth and development. A dramatic example is the reprogramming of promoter specificity of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) after infection with phages such as SP01 or SP82 in which a viral protein replaces the normal B. subtilis a' subunit (1-3). This type of regulatory mechanism has long been considered an attractive possibility in normal cells (4); however, although many "altered" forms of RNA polymerase have been reported (refs. 5-11, see refs. 12-14 for reviews), it is only quite recently that bacterial RNA polymerases have been detected that clearly express novel promoter specificities (15,16 MATERIALS AND METHODSExperimental procedures and materials have been described previously (16) except where noted. Growth ofB. subtilis W168 in early exponential phase and preparation of fraction 5 RNA polymerase proceeded as before (16) except that: (i) precipitation of RNA polymerase with ammonium sulfate employed 0.4 g/ml after Polymin fractionation and 0.42 g/ml after DNA cellulose chromatography; (ii) the precipitate from the first ammonium sulfate fractionation was desalted by chromatography on Bio-Gel P-10 (Bio-Rad) in a buffer containing 10 mM Tris-HCl (pH 8), 1 mM EDTA, 1 mM dithiothreitol, 10% (vol/ vol) glycerol, and 50 mM NaCl; (iii) RNA polymerase was eluted from the DNA cellulose column with a 4 column volume gradient (0.05-1.0 M NaCl).B. subtilis core RNA polymerase was prepared by chromatography of peak fractions of RNA polymerase holoenzyme I from heparin agarose (fractions HA 116-132) on phosphocellulose (21) followed by rechromatography on poly(rC)-cellulose (22) to remove a contaminating polypeptide of Mr 92,000. RNA polymerase from the phosphocellulose column (1.5 mg) in 10 mM Tris-HCl (pH 8)/1 mM EDTA/10 mM 2-mercaptoethanol/ 10 mM MgCl2/10% (vol/vol) glycerol/0.02 M NaCl was passed through a 2-ml poly(rC)-cellulose column. Flow-through fractions were pooled with those eluted with 0.1 M NaCl and were concentrated by dialysis against 50% (vol/vol) 2762The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

“…Some classes of middle and late phage genes can be transcribed in the absence of ai (28,29). It should be possible to confirm that coliphage T4 late genes are expressed without a (29) by using strain 285c and shifting to nonpermissive temperature after T4 early genes have been expressed.…”

Section: Discussionmentioning

confidence: 99%

Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase.

Harris¹,

Heilig²,

Martinez³

et al. 1978

A gene affecting the or subunit of DNA-dependent RNA polymerase is tightly linked to dnaG at 66 min on the Escherichia coli chromosome. In order to create an easily selectable marker in this region, we inserted transposon-10, which carries a gene determining resistance to tetracycline (tet) near 66 minm and the order toC-dnaG--tet was determined. We used frequency of cotransduction with tet as a criterion to screen a collection of spontaneous temperature-sensitive Escherichia coli mutants that might affect the a subunit. One such mutant was found to map at the or locus. The a subunit isolated from this mutant is unstable at 460C in vitro and has an altered electrophoretic mobility. The temperature sensitivity of RNA synthesis in this mutant indicates that most transcription in E. coli is cr dependent.The a subunit of Escherichia coli DNA-dependent RNA polymerase is responsible for much of the specificity of in vitro transcription (1, 2). For example, RNA polymerase core enzyme (lacking a subunit) binds weakly to many sites on T7 phage DNA in vitro, whereas RNA polymerase holoenzyme (containing a subunit) binds strongly and only to those sites at which in vivo transcription begins (3).Elucidation of the role of the a protein in the specificity of gene expression has been delayed, however, by the lack of a subunit mutants. Previous genetic studies on the electrophoretic differences among the a proteins of enteric bacteria suggested that the structural gene for a maps at about 66 min on the E. coli genome (4, 5). In order to rapidly screen potential a mutants in this region of the genome, we have inserted the translocatable tetracycline-resistance (tet) element TnlO near the a gene. By using this readily selectable marker for rapid mapping of mutants, we have identified a spontaneous temperature-sensitive (ts) mutant that produces an altered a protein and is deficient in the synthesis of RNA at nonpermissive temperatures.MATERIALS AND METHODS Media, Buffers, and Chromatographic Materials. LB broth (6) supplemented with 0.4% glucose was used; the NaCl concentration was adjusted to 0.1 M. L plates consist of LB broth and 2% Difco agar. LC plates contain, in addition, 5 mM CaCd2 but 1.5% agar. Davis minimal medium is described by Calendar and Lindahl (7). Buffer P50 is described by Gonzalez et al. (8) and TGED buffer is described by Burgess and Jendrisak (9). TPG-CAA medium (10) enriched with 0.8% glucose, 0.01% arginine, 0.5 mM CaCI2, and 20 amino acids and trace metals each at 1 ttg/ml was used for radioactive labeling. Bio-Gel A-1.5m used for gel filtration and the ion-exchange material Bio-Rex 70 were purchased from Bio-Rad. (11), selecting for tetracycline-resistance and the ability to grow at 42'C (dnaG +). This procedure yielded transductants with a tet gene that is highly cotransducible with dnaG. C-2359 is one of these.Purification of RNA Polymerase. RNA polymerase holoenzyme was purified according to the procedure of Burgess and Jendrisak (9). After the enzyme was eluted from the A-1.5m column, i...