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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We identified and sequenced the gene for the Chlamydia trachomatis RNA polymerase major sigma subunit. The gene encodes a 66,141-dalton protein (sigma 66), intermediate in size between the major sigma subunits of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The C. trachomatis sigma 66 subunit had extensive amino acid homology with the sigma 70 and sigma 43. The sigma subunit regions purportedly involved in core enzyme binding and DNA promoter recognition were also highly conserved, despite the lack of a DNA promoter consensus sequence between E. coli and C. trachomatis promoters and the inability of E. coli holoenzyme to specifically transcribe chlamydial genes. Compared with E. coli sigma 70, there were some major differences in the chlamydial sigma 66 sequence, including a gap of 63 amino acids and an additional 16 amino acids at the carboxyl terminus, which may play some role in modifying the sigma-DNA interaction, such that a promoter sequence unique to C. trachomatis is recognized. Monoclonal antibodies specific for E. coli sigma 70 were used to probe for homologous structures between sigma 70 and sigma 66; only one of seven antibodies bound specifically to sigma 66, suggesting minimal conservation of antigenic sites. The chlamydial sigma 66 was present in elementary bodies and was expressed throughout the developmental cycle, which implied that this gene encodes the major vegetative sigma subunit. Because the ability to study the genetics of C. trachomatis is currently limited, this work provides a tool for more detailed study of chlamydial promoter structure and of coordinate gene expression during the developmental cycle.
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PMID:Chlamydia trachomatis RNA polymerase major sigma subunit. Sequence and structural comparison of conserved and unique regions with Escherichia coli sigma 70 and Bacillus subtilis sigma 43. 214 44

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.
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PMID:Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction. 221 7

We analysed transcription of the DNA region immediately downstream of the origin of replication in the chlamydial plasmid pCT. This region comprises two convergent open reading frames (ORF7, ORF8), encoding putative polypeptides that are homologous to each other and with C-terminal domains typical of the phage integrase family of proteins. Northern blot and RNA 5' end mapping analyses indicated that both ORFs were transcribed in the late phase of the chlamydial replicative cycle. RNA mapping showed the presence of a transcript starting 31 nucleotides (nt) before the ATG start codon of ORF7, and two temporally regulated transcripts starting 59 and 89 nt upstream of the ATG start codon of ORF8. Two abundant RNA species of 225 and 415 nt were also identified as overlapping anti-sense transcripts (AS-RNAs), complementary to the 3' end of ORF8 mRNA, with identical 5' ends but different 3' ends. In vitro and in vivo experiments in Escherichia coli showed that the sigma 70-RNA polymerase complex was capable of initiating RNA synthesis at the same sites as observed in Chlamydia trachomatis for ORF7 and AS-RNA transcripts, but was not able to transcribe ORF8. In accord with this, sequences at -10 and -35 nt upstream of the RNA 5' ends resemble sigma 70 consensus promoters in the case of ORF7 and AS, but not in the case of the two ORF8 transcripts. Therefore, transcription of ORF7 and ORF8 is controlled by different types of promoters.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transcriptional analysis of the Chlamydia trachomatis plasmid pCT identifies temporally regulated transcripts, anti-sense RNA and sigma 70-selected promoters. 768 69

We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.
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PMID:Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis. 773 Feb 99

The complete nucleotide sequence of the rpoB gene which encodes the beta subunit of S. aureus RNA polymerase has been determined. The RpoB protein, consists of 1182 amino acids and has a novel initiation codon UUG which initiates protein synthesis with methionine. There is a very strong Shine-Dalgarno complementarity and the -10 and -35 promoter hexameric sequences are TAATAT and CCGTTT, respectively. A rho-dependent termination site, CAATCAA, is present which overlaps the -35 promoter sequence of the adjacent rpoC gene a feature which may have a role in the co-ordinate expression of the two genes. A strong homology and conserved regions were found to exist over the predicted amino acid sequences coding for S. aureus rpoB and the equivalent proteins in Escherichia coli, Pseudomonas putida, Salmonella typhimurium, Chlamydia trachomatis, cyanobacterium Anabaena sp. strain PCC 7120.
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PMID:Nucleotide sequence of the Staphylococcus aureus RNA polymerase rpoB gene and comparison of its predicted amino acid sequence with those of other bacteria. 777 3

Obligate parasitic bacteria of the genus Chlamydia possess a developmental cycle that takes place entirely within eucaryotic host cells. Because standard methods of genetic analysis are not available for chlamydiae, an in vitro transcription system has been developed to elucidate the mechanisms by which chlamydiae regulate gene expression. The in vitro system is specific for chlamydial promoters but is inefficient, presumably because the RNA polymerase is not saturated with sigma factor. Therefore, we prepared recombinant Chlamydia psittaci 6BC major sigma factor to enhance transcription in the in vitro system. The gene encoding the major sigma factor (sigA) was identified by using an rpoD box oligonucleotide and was subsequently cloned and sequenced. It was found to encode a potential 571-amino-acid protein (sigma 66) that is greater than 90% identical to the previously identified major sigma factors from the L2 and MoPn strains of Chlamydia trachomatis. sigA was recloned into a T7 RNA polymerase expression system to produce large quantities of sigma 66 in Escherichia coli. Overexpressed sigma 66 was identified by immunoblot by using monoclonal antibodies 2G10 (reactive) and 2F8 (nonreactive) generated against E. coli sigma 70. After purification by polyacrylamide gel electrophoresis, the recombinant protein was found to stimulate, by 10-fold or more, promoter-specific in vitro transcription by C. psittaci 6BC and C. trachomatis L2 RNA polymerases. Transcription was dependent on added chlamydial sigma 66, rather than on potentially contaminating E. coli sigma 70 or other fortuitous activators, since the monoclonal antibody 2G10, and not 2F8, inhibited transcription initiation. Recombinant omega(66) had no effect on transcription by E. coli core polymerase. The addition of recombinant omega(66) to the in vitro system should be useful for distinguishing omega(66)-dependent transcription of developmentally regulated chlamydial genes from omega(66)-independent transcription.
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PMID:Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC. 802 Dec 4

Mutated variants of the predicted promoter of the countertranscript of the Chlamydia trachomatis plasmid were tested by in vitro transcription with chlamydial extract. A 3-bp deletion within the -10 region of the putative promoter caused the RNA polymerase to initiate transcription 3 bases downstream. Many single mutations in the -10 and -35 regions did not alter promoter function. However, some multiple mutations in both hexamers rendered the promoter inefficient or ineffective. Taken together, these results indicate that (i) the sequence requirement for chlamydial promoters differs from that for Escherichia coli and (ii) chlamydial RNA polymerase can tolerate considerably more variation at the -10 and -35 regions. These results are paradoxical considering the homology between C. trachomatis sigma 66 and E. coli sigma 70.
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PMID:The RNA polymerase of Chlamydia trachomatis has a flexible sequence requirement at the -10 and -35 boxes of its promoters. 820 57

We have cloned the chlamydial operon that encodes the initiation factor IF1, the ribosomal proteins L36, S13, and S11, and the alpha subunit of RNA polymerase. The genes for S11 and alpha are closely linked in Escherichia coli, Bacillus subtilis, and plant chloroplast genomes, and this arrangement is conserved in Chlamydia spp. The S11 ribosomal protein gene potentially encodes a protein of 125 amino acids with 41 to 42% identity over its entire length to its E. coli and B. subtilis homologs; the gene encoding the alpha subunit specifies a protein of 322 amino acids with 25 to 30% identity over its entire length to its E. coli and B. subtilis homologs. In a T7-based expression system in E. coli, the chlamydial alpha gene directed the synthesis of a 36-kDa protein. Mapping of the chlamydial mRNA transcript by RNase protection studies and by a combination of reverse transcription and the polymerase chain reaction demonstrates that IF1, L36, S13, S11, and alpha are transcribed as a polycistronic transcript.
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PMID:Cloning and characterization of the RNA polymerase alpha-subunit operon of Chlamydia trachomatis. 822 62

The gene encoding the Chlamydia trachomatis histone H1-like protein (Hc1) from serovar L2 was cloned into Escherichia coli by use of expression vector pET11d. In this vector, transcription of the gene is under the control of a bacteriophage T7 promoter, and T7 RNA polymerase is inducible in the host. Following induction, the E. coli cells were lysed gently. Gel filtration of the lysate revealed comigration of DNA and Hc1 in the voided volume. Electron microscopy revealed the DNA to be complexed with protein in large aggregates, often in the form of spherical bodies. Purified recombinant Hc1 maintained its DNA-binding capacity and was able at high concentrations to form condensed aggregates with DNA (one molecule of Hc1 per base pair) independently of the form or size of the DNA but with a slight preference for supercoiled DNA. Hc1 alone is thus able to package DNA into condensed spherical bodies.
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PMID:Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA. 844 85

Extracts of Chlamydia psittaci and Chlamydia trachomatis were used to transcribe molecularly cloned chlamydial genes in vitro. The extracts were prepared by lysing reticulate bodies, obtaining the 10,000 x g centrifugation pellet, and eluting RNA polymerase from the pellet by treatment with 2M KCl to yield a fraction designated SS2. Some in vitro transcription was initiated from non-chlamydial promoters and a small amount of transcription was from endogenous DNA template in SS2. However, optimal transcription from exogenous templates required chlamydial promoter sequences, and primer extension analysis indicated that chlamydia promoter-specific in vitro transcription was initiated from the same start sites recognized in vivo. A monoclonal antibody that was generated against Escherichia coli sigma 70 and which immunologically cross-reacts with C. trachomatis sigma 66 inhibited in vitro transcription of vector and cloned chlamydial DNA, suggesting that transcriptional initiation in the SS2 fraction is mediated by sigma 66. An in vitro transcription assay based on detection of transcripts of specific lengths was applied to the chlamydial system; this assay and others described here should be useful in defining chlamydial promoters and other transcriptional regulatory elements.
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PMID:In vitro transcription in Chlamydia psittaci and Chlamydia trachomatis. 848 21


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