RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography

Plevani, Paolo; AIbertini, Alessandra M.; Galizzi, Alessandro; Adamoli, Anna; Mastromei, Giorgio; Riva, Silvano; Cassani, Giovanni

doi:10.1093/nar/4.3.603

Cited by 23 publications

(8 citation statements)

References 28 publications

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“…enzyme-DNA complexes resistant to the antibiotic during the preincubation time, similar to what has been shown for bacterial ribonucleic acid polymerase and rifampin (11,18).…”

Section: Ir1-i0 -1 Nosupporting

confidence: 73%

Effect and mechanism of action of aphidicolin on yeast deoxyribonucleic acid polymerases

Plevani¹,

Badaracco²,

Ginelli³

et al. 1980

Antimicrob Agents Chemother

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The antibiotic aphidicolin inhibited in vitro deoxyribonucleic acid synthesis catalyzed by crude yeast extracts and by partially purified yeast deoxyribonucleic acid polymerases. The mechanism of action of aphidicolin on yeast deoxyribonucleic acid polymerase I was noncompetitive with deoxyguanosine 5'-triphosphate, deoxyadenosine 5'-triphosphate, and deoxythymidine 5'-triphosphate and was of the mixed type with deoxycytidine 5'-triphosphate. The relative ratio of enzyme to the template-initiator complex was important for detecting the inhibitory effect of the antibiotic. The inhibition of in vitro deoxyribonucleic acid synthesis by aphidicolin was reversible, and the effect on yeast deoxyribonucleic acid polymerase might have been partially mediated by some supplementary factor(s).Aphidicolin, a tetracycline diterpenoid produced by the mold Cephalosporium aphidicola (5), inhibits cellular deoxyribonucleic acid (DNA) synthesis and the growth of herpes simplex and vaccinia viruses (6). Among in vivo macromolecular syntheses, only DNA synthesis is sensitive to the drug (14). A differential effect of the antibiotic on the three DNA polymerases from eucaryotic sources has recently been shown; aphidicolin specifically inhibits DNA polymerase a, whereas DNA polymeras I8 and y are not inhibited (14,17 Yeast strain and purification of yeast DNA polymerases. Saccharomyces cerevisiae strain 6175/ lla a nut-2 from Istituto di Genetica, Universita di Milano, Italy, was grown to the late logarithmic phase, harvested, and stored as previously described (19). Yeast DNA polymerases were purified as described by Chang (9) from a frozen yeast cell pellet (equivalent to 80 g) up to the phosphocellulose chromatography step (9). The active fractions were pooled, dialyzed against 25 mM potassium phosphate (pH 7.2) in 5 mM 2-mercaptoethanol-10% glycerol, and loaded onto a Whatman diethylaminoethyl 32 cellulose column (1.5x 10 cm) previously equilibrated with the same buffer. The column was washed and then eluted with a 200-ml linear gradient of 0 to 0.42 M KCl in 25 mM potassium phosphate (pH 7.2)-5 mM 2-mercaptoethanol-10% glycerol. Two poorly resolved peaks of activity were obtained from this column. Active fractions eluting between 0.06 and 0.13 M KCl (fraction I) and active fractions eluting between 0.22 and 0.30 M KCl (fraction II) were pooled separately; they correspond to yeast DNA polymerases I and II, respectively (9). Proteins in fractions I and II were precipitated by dialysis versus ammonium sulphate (60% saturation) in 50 mM potassium phosphate (pH 7.2)-5 mM 2-mercaptoethanol. The precipitated proteins were collected by centrifugation and dissolved in 50 mM potassium phosphate (pH 7.2)-5 mM 2-mercaptoethanol-10% glycerol (

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“…enzyme-DNA complexes resistant to the antibiotic during the preincubation time, similar to what has been shown for bacterial ribonucleic acid polymerase and rifampin (11,18).…”

Section: Ir1-i0 -1 Nosupporting

confidence: 73%

Effect and mechanism of action of aphidicolin on yeast deoxyribonucleic acid polymerases

Plevani¹,

Badaracco²,

Ginelli³

et al. 1980

Antimicrob Agents Chemother

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“…The RNAP holo and core enzymes, isolated from E. coli strain MRE‐600 (ATCC 29417; ATCC), were checked for the presence and absence of the σ subunit by SDS/PAGE. Bacillussubtilis RNAP was a kind gift of A. Galizzi (Institute of Genetics, University of Pavia, Italy) [20]. Rifampicin‐resistant (rif R ) E. coli RNAP (rpoB3) was from Promega (Madison, WI, USA); rif R RNAP (rpoB7) and rif R RNAP (rpoB3595) were purified, respectively, from E. coli strains CAG3516 and CAG3595 [21], following the purification procedure described previously [22].…”

Section: Enzymes and Antibioticsmentioning

confidence: 99%

Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase

Sarubbi

Monti

Corti

et al. 2004

European Journal of Biochemistry

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GE23077, a novel microbial metabolite recently isolated from Actinomadura sp. culture media, is a potent and selective inhibitor of bacterial RNA polymerase (RNAP). It inhibits Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) RNAPs with IC 50 values (i.e. the concentration at which the enzyme activity is inhibited by 50%) in the 10 )8 M range, whereas it is not active on E. coli DNA polymerase or on eukaryotic (wheat germ) RNAP II (IC 50 values > 10 )4 M in both cases). In spite of its potent activity on purified bacterial RNAPs, GE23077 shows a narrow spectrum of antimicrobial activity on Gram-positive and Gram-negative bacteria. To investigate the molecular basis of this behaviour, the effects of GE23077 on macromolecular biosynthesis were tested in E. coli cells permeabilized under different conditions. The addition of GE23077 to plasmolyzed cells resulted in an immediate and specific inhibition of intracellular RNA biosynthesis, in a dose-response manner, strongly suggesting that cell penetration is the main obstacle for effective antimicrobial activity of the antibiotic. Biochemical studies were also conducted with purified enzymes to obtain further insights into the mode of action of GE23077. Interestingly, the compound displays a behaviour similar to that of rifampicin, an antibiotic structurally unrelated to GE23077: both compounds act at the level of transcription initiation, but not on the r subunit and not on the formation of the promoter DNA-RNAP complex. Tests on different rifampicin-resistant E. coli RNAPs did not show any cross-resistance between the two compounds, indicating distinct binding sites on the target enzyme. In conclusion, GE23077 is an interesting new molecule for future mechanistic studies on bacterial RNAP and for its potential in anti-infective drug discovery.Keywords: antibiotic; cell permeabilization; natural product; rifampicin; transcription initiation. DNA-directed RNA polymerase (EC 2.7.7.6; RNAP) is the central enzyme of bacterial gene expression, responsible for all cellular RNA synthesis [1]. The catalytically competent ÔcoreÕ RNAP consists of five subunits (a 2 bb¢x, with a combined molecular mass of % 400 kDa) and is capable of elongation and termination. The initiation-competent ÔholoÕ RNAP is composed of the core enzyme and of an additional subunit, r, which confers on RNAP the ability to initiate transcription at specific promoter sites [2,3]. After over four decades of intensive research, RNAP is currently the subject of renewed interest and excitement, owing to recent publication of the crystal structures of the core [4] and holo [5,6] enzymes, and of an RNAP-DNA complex [7].The transcription process consists of three main stages: initiation, elongation and termination. Transcription initiation is a multistep process [8] in which holo RNAP specifically binds to promoter DNA at positions )35 and )10 to form an RNAP-promoter closed complex, melts the DNA duplex around the )10 region to yield an RNAPpromoter open complex, and then initiates transc...

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“…A is the principal sigma factor present in vegetatively growing B. subtilis. E-A was the first holoenzyme isolated from B. subtilis, being readily purified by the standard chromatographic techniques that had been used for the isolation of the E. coli enzyme (8,185,190,234,270,271). A , like its E. coli counterpart 70 , is separable from core RNAP by chromatography on phosphocellulose (190,271).…”

Section: Isolation and Characterizationmentioning

confidence: 99%

The sigma factors of Bacillus subtilis.

Haldenwang¹

1995

Microbiological Reviews

368

258

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RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography

Cited by 23 publications

References 28 publications

Effect and mechanism of action of aphidicolin on yeast deoxyribonucleic acid polymerases

Effect and mechanism of action of aphidicolin on yeast deoxyribonucleic acid polymerases

Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase

The sigma factors of Bacillus subtilis.

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