The nudeotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E. coil K12 (original) (raw)
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Proceedings of the National Academy of Sciences of the United States of America, 1979
A transducing phage has been isolated which codes for the a subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage X vector that is capable of forming Iyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the a gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and o (rpoD) whereas those containing the Sac I fragment complement only a. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/ translation system suggest that a Sac I site separates the promoter for a from the a structural gene. The direction of transcription of a was determined to be clockwise with respect to the E. coli genetic map. Escherichia coli RNA polymerase is a multisubunit enzyme composed of a, 13, f3', and oa subunits. The enzyme is found in two forms: as holoenzyme (a2/3f'), capable of selective DNA
Altered chemical properties in three mutants of E. coli RNA polymerase sigma subunit
Molecular and General Genetics MGG, 1979
We have analyzed some chemical properties of the sigma subunit of RNA polymerase from the sigma mutants: rpoD1 (Gross et al., 1978), rpoD2 (formerly known as a/t-l) (Silverstone et al., 1972; 1"ravers et al., 1978), and rpoD800 (Gross et al., 1979). Each of the three mutants is located at about 66 min on the E. coli genetic map and exhibits an alteration in the enzymatic properties of its sigma subunit. The tryptic peptides and isoelectric focusing behavior were analyzed for mutant and wild type sigma. A single, but different altered lysine tryptic peptide was observed for each mutant. No altered arginine tryptic peptides were observed. The rpoD800 mutant sigma showed an altered isoelectric point. These studies provide chemical evidence that the sigma polypeptide in all three mutants is altered and strongly support the conclusion that the mutations are in the structural gene for sigma.
Proceedings of the National Academy of Sciences, 1979
The genes for the RNA polymerase a subunit (rpoD) and DNA primase (dnaG) of Salmonella typhimurium have been cloned into X vectors. Combined restriction, deletion, and functional analysis of the cloned fragment allows us to map the genes precisely on the fragment, establishes the direction in which rpoD is transcribed, and reveals the existence of at least one new gene in the vicinity. A closely homologous, smaller fragment of Escherichia coli DNA, also cloned into X, contains rpoD and at least part of dnaG.
European Journal of Biochemistry, 1981
The combined structural study of proteins and of their corresponding genes utilizing the methods of both protein and nucleotide chemistry greatly accelerates and considerably simplifies both the nucleotide and protein structure determination and, in particular, enhances the reliability of the analysis. This approach has been successfully applied in the primary structure determination of the fl and j' subunits of Escherichia coli DNAdependent RNA potynierase and of their structural genes, yielding a continuous nucleotide sequence (4714 base pairs) that embraces the entire rpoB gene, the initial part of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the fl subunit, comprising 1342 residues, and the N-terminal sequence of the jl' subunit (1 76 residues).
Molecular and General Genetics MGG, 1980
We have characterized a new mutation rpoD800 affecting the sigma gene of E. coli. Upon transfer to high temperature, a strain with the rpoD800 mutation ceases growth within 30 min. We find that this mutation renders sigma about 10-fold more thermolabile than the wild type sigma at 45 ° C in vitro. We have compared the temperature profile for inactivation of wild type and mutant sigma and find that the mutant inactivates at a temperature about 9 ° C lower than does the wild type. The chromosomal locus affected by rpoD800 is shown to be allelic to the locus affected by the spontaneous mutants ts285 and alt-1. All three mutations result in altered sigma and in altered growth at high temperature. We argue that the single locus affected is the structural gene for the sigma subunit of E.
Cell, 1983
The sigma subunit of E. coli RNA polymerase is encoded by the rpoD gene. Within the sequence upstream from rpoD, we have identified the structural genes rpsU and dnaG, which encode the 30s ribosomal protein 521 and DNA primase, respectively. The three genes are in the order rpslJ, dnaG rpoD, and are all encoded by the same DNA strand. Analysis of in vivo transcripts from this region shows that these genes are all within the same operon. By correlating the 5' and 3' ends of in vivo transcripts with our DNA sequence, we have identified several regulatory features of the operon. These features include tandem promoters upstream from rpsll, a terminator between rpsUand dnaG, an RNA processing site separating dnaGand rpoD, and the operon terminator just downstream from rpoD. Immediately upstream of the operon promoters is an active promoter for an unidentified gene. We discuss the regulatory significance of the operon features and the biological significance of an operon encoding proteins essential for translation, replication and transcription.
A general view: Structure and function of the subunits of E. coli RNA polymerase
Journal of Cell and Molecular Biology, 2003
The DNA-dependent RNA polymerases are widespread throughout nature. E. coli RNA polymerase, one of the most well characterized polymerase, consists of two major forms, core enzyme with subunit stoichiometry of α 2 ββ' and holoenzyme which contains an additional σ subunit to core enzyme. E. coli RNA polymerase plays a central role in transcription. While the core enzyme catalyses the elongation and termination of transcription, to initiate core enzyme needs to combine with σ subunit. The three dimensional structure of this multimeric enzyme revealed a thumb-like projection. Using the electron microscope, Tichelar and Heel (1990) proposed a model that is in agreement with both β and β' together constituting a V-like structure and α dimer associates at the short ends, while σ is positioned within the concave side of the core, next to the dimer. In this review, the structure and related functions of the subunits of E. coli DNA-dependent RNA polymerase is presented based on several researches and reviews. Considering biochemical and genetic studies on the RNA polymerase of E. coli, a genetic walk on the subunits is summarized.
Microbiology, 1994
The rpoN gene (encoding the sigma factor a") of Escherichia coli was cloned and its nucleotide sequence determined. Promoter probe analysis confirmed the presence of a promoter in a 350 bp fragment covering the start of rpoN. The likely promoter was identified. The nucleotide sequence of the region extending 2.1 kb downstream of rpoN was also determined. This region contained four open reading frames encoding potential polypeptides of 10750, 17959,32492 and 9810 Da; maxicell and T7 promoter studies showed that four polypeptides of similar molecular masses were expressed from this region. The amino acid sequence of the 17959 Da polypeptide showed homology to the enzyme IIA domains of several proteins of the bacterial sugar phosphotransferase system (PTS), and the 981 0 Da polypeptide showed homology to the HPr proteins of the bacterial PTS. The proteins encoded downstream of rpoN are known to negatively regulate 6" activity. The homologies therefore suggest that this effect on t~~~ may be mediated by sequential protein phosphorylation and suggest that there is a link between signal transduction and transcription of 0"-dependent genes.
Proceedings of the National Academy of Sciences, 1995
We present a simple, rapid procedure for reconstitution ofEscherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant a, ,B, (', and a7O subunits. Hexahistidine-tagged recombinant a subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant 13, 13', and cr70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, a-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with a(1-235), an a subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.