A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase

Gonzàlez, Nèlida N.; Wiggs, Janey L.; Chamberlin, Michael J.

doi:10.1016/0003-9861(77)90521-5

Cited by 305 publications

(128 citation statements)

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“…The coimmunoprecipitation of FirA by RNA polymerase antiserum reported here is suggestive of such an interaction and strengthens the hypothesis that FirA is directly involved in transcription. However, numerous studies (5,6,10,30) have shown that only the %23cr holoenzyme complex is required for in vitro transcription. Possibly FirA is required catalytically for certain phases of growth or only under certain growth conditions.…”

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Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli

Dicker

Seetharam

1991

J Bacteriol

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The Escherichia coli genefirA, previously reported to code for a small, histonelike DNA-binding protein, has been cloned and found to reside immediately downstream from skp, a gene previously identified as the firA locus. firA encodes a 36-kDa protein. The mutantfirA200(Ts) allele was also cloned and shown to contain three mutations, each mutation giving rise to a single amino acid change. Partially purified wild-type FirA It is well documented that selection for rifampin resistance in Escherichia coli gives mutations in rpoB, the gene encoding the P subunit of RNA polymerase (13,16,27). Suppressors of the rifampin resistance phenotype are worth studying because of the insight they might provide into the components of the transcription machinery. Several loci which can suppress this rifampin resistance have been identified and have been termed fir mutants (rif spelled backwards): firA (19) in the 4-min region, firB (26) in the 90-min region, and firC (3). Among these, firA has been the most studied. The firA 200(Ts) allele has been shown to cause temperature sensitivity for growth and to reverse the rifampin resistance of rifampin-resistant rpoB mutants (21). firA was also reported to confer temperature sensitivity to in vitro RNA synthesis by RNA polymerase partially purified from a firA200(Ts) strain and has been hypothesized to interact with RNA polymerase in vivo (22). A recombinant lambda phage containing a 6.7-kb insert was shown to complement the firA200(Ts) phenotypes (20) In this study, we report the cloning of thefirA and firA200 genes and show thatfirA is located immediately downstream from the previously reported skp gene. We also discuss data which indicate that the FirA protein coimmunoprecipitates with RNA polymerase holoenzyme. This study is part of our larger interest in better defining the role, if any, of firA in transcription.MATERIALS AND METHODS Strains and media. All strains used are listed in Table 1. Strain RL25-1 was obtained from R. Menzel (Bristol-MyersSquibb Co.), and KK2186 (31) was a gift from R. Zagursky (Du Pont Co.). JCR20, containing the authentic firA200(Ts) allele, was kindly supplied by J. Coleman, University of Louisiana, and was obtained by crossing a P1 lysate of the authentic RL25 into MF6R (Rif' dapD) (7) and selecting for * Corresponding author.prototrophs. The library of TnJOd-Cam transposon insertions was made (A. Segall, National Institutes of Health) by the reference method (9). Cells were grown in LB medium alone or LB medium containing 150 ,ug of ampicillin per ml or 150 or 50 ,ug of rifampin per ml. For maintenance of pMS421, spectinomycin was used at 50 p,g/ml. ID15 was isolated as a spontaneous rifampin-resistant colony on LB plates containing 150 pug of rifampin per ml. Plates were incubated at 42 and 43°C in water-jacketed incubators.Strain construction. P1 transduction was performed as previously described (24)

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Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli

Dicker

Seetharam

1991

J Bacteriol

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“…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.…”

Section: Methodsmentioning

confidence: 99%

“…These fractions were diluted with TGED buffer (8) to a salt concentration of 0.1 M and concentrated on small DEAE-cellulose columns (1 ml of packed volume per 10 mg of protein). Each enzyme fraction was eluted from the DEAEcellulose in TGED buffer containing 0.5 M NaCI, diluted, and further purified on a Bio-Rex 70 column (8). The core enzyme and a subunit fractions were concentrated and stored as described by Burgess and Jendrisak (9 Some of the mapping of the a gene was done with two rethe 66min region of the E. colt chromosome the gene order is cently isolated a mutants.…”

Section: Methodsmentioning

confidence: 99%

“…The RNA polymerase holoenzyme and core enzyme were then eluted as described (9). These fractions were diluted with TGED buffer (8) to a salt concentration of 0.1 M and concentrated on small DEAE-cellulose columns (1 ml of packed volume per 10 mg of protein). Each enzyme fraction was eluted from the DEAEcellulose in TGED buffer containing 0.5 M NaCI, diluted, and further purified on a Bio-Rex 70 column (8).…”

Section: Introductionmentioning

confidence: 99%

“…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, it was concentrated and loaded onto a phosphocellulose column as described by Gonzalez et al (8). The RNA polymerase holoenzyme and core enzyme were then eluted as described (9).…”

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

Harris¹,

Heilig²,

Martinez³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Facilitated diffusion of a DNA binding protein on chromatin.

Hannon¹,

Richards²,

Gould³

1986

The EMBO Journal

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Facilitated diffusion accounts for the rapid rate of association of many bacterial DNA binding proteins with specific DNA sequences in vitro. In this mechanism the proteins bind at random to non‐specific sites on the DAN and diffuse (by ‘sliding’ or ‘hopping’) along the DNA chain until they arrive at their specific functional sites. We have investigated whether such a mechanism can operate in chromatin by using a bacterial DNA binding protein, Escherichia coli RNA polymerase, that depends on linear diffusion to locate initiation sites on DNA. We have measured the competition between chromatin and its free DNA for the formation of initiation complexes. Only the short linker segments exposed by the removal of histone H1 are available for interaction with the polymerase, but the sparsely distributed promoter sites on the linker DNA of such a polynucleosome chain are located at the same rate as those on DNA. We conclude that the polymerase is free to migrate between the separate linker DNA segments of a polynucleosome chain to reach a promoter site. This chain thus permits the ‘hopping’ of proteins between neighboring linker segments in their search for a target site on the accessible DNA.

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A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase

Cited by 305 publications

References 15 publications

Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli

Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli

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

Facilitated diffusion of a DNA binding protein on chromatin.

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