Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

A long-standing question in the biology of the intracellular bacterium, Chlamydia, has been the structure of the promoter recognized by its RNA polymerase. The 'RNA polymerase sigma subunit paradox' refers to the difficulty reconciling the conservation between the RNA polymerases of Chlamydia and Escherichia coli, especially at the level of the promoter-recognition sigma subunit, with the general lack of homology between chlamydial promoters and the E.coli sigma(70) consensus promoter. While the -10 promoter element appears to be conserved between Chlamydia and E.coli, the structure of the chlamydial -35 promoter element has not been defined. We have investigated the structure of the -35 element of the Chlamydia trachomatis dnaK promoter by measuring the effects of single base pair substitutions on in vitro promoter activity. Most substitutions produced large decreases in promoter activity, which allowed us to define the optimal -35 sequence in the context of the dnaK promoter. We found that the optimal chlamydial -35 promoter sequence is identical to the E.coli sigma(70) consensus -35 promoter element (TTGACA). These results indicate that the optimal promoter specificities of the major form of chlamydial RNA polymerase and E.coli sigma(70) RNA polymerase are in fact highly conserved. A further implication of our results is that many chlamydial promoters have a suboptimal promoter structure. We hypothesize that these chlamydial promoters are intrinsically weak promoters that can be regulated during the chlamydial developmental cycle by additional transcription factors.
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PMID:Mutational analysis of the Chlamydia trachomatis dnaK promoter defines the optimal -35 promoter element. 1252 61

Chlamydia is predicted to encode two alternative sigma factors that could provide a mechanism for the regulation of gene expression via alternative forms of RNA polymerase. We have demonstrated that sigma 28, one of these alternative sigma factors, is transcriptionally active. Chlamydial sigma 28 RNA polymerase was reconstituted from recombinant sigma 28 protein and core enzyme that was biochemically isolated from chlamydiae. In an in vitro transcription assay, sigma 28 RNA polymerase transcribed the hctB promoter in a sigma 28-dependent manner. Transcription by sigma 28 RNA polymerase was salt tolerant compared with transcription by sigma 66 RNA polymerase, the major form of chlamydial RNA polymerase. As hctB encodes a histone-like protein that is only expressed late in the developmental cycle, our results suggest that sigma 28 RNA polymerase has a role in the regulation of late gene expression in Chlamydia.
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PMID:Sigma28 RNA polymerase regulates hctB, a late developmental gene in Chlamydia. 1461 80

The rpsD gene of Chlamydia trachomatis encodes the alternative sigma factor sigma28, which bears strong homology to many bacterial sigma factors, including Escherichia coli sigma8 and Bacillus subtilis sigmaB and sigmaD. Recently, a sigma28 promoter was identified upstream of the late-cycle-expressed gene hctB, which encodes the Chlamydia-histone-like protein 2 (Yu & Tan, 2003). In this study it is shown that the product of chlamydial rpsD is an E. coli sigma28 homologue. It was found that recombinant chlamydial sigma8, in combination with E. coli core RNA polymerase, initiates transcription in vitro from the E. coli sigma28-dependent promoter of fliC. It was also demonstrated that the recombinant chlamydial sigma28 does not recognize major sigma factor sigma70-consensus-like sequences in vitro. In C. trachomatis-infected cells, two rpsD transcripts were detected with 5' ends located 18 (transcript I) and 54 bp (transcript II) upstream of the translational initiation codon at 16 and 30 h post-infection. When the temperature of cultures infected with C. trachomatis was shifted from 35 to 42 degrees C, the rpsD transcript I increased dramatically. The levels of chlamydial sigma28, relative to EF-Tu, were greater throughout the exponential growth phase of the reticulate body, but lower late in the developmental cycle. These data support the hypothesis that sigma28 plays a role in the regulatory network that allows chlamydiae to survive changes in its environment, enabling it to complete its unique developmental cycle.
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PMID:Chlamydia trachomatis sigma28 recognizes the fliC promoter of Escherichia coli and responds to heat shock in chlamydiae. 1470 14

Comparative analysis of the domain architectures of the beta, beta', and sigma(70) subunits of bacterial DNA-dependent RNA polymerases (DdRp), combined with sequence-based phylogenetic analysis, revealed a fundamental split among bacteria. DNA-dependent RNA polymerase subunits of Group I, which includes Proteobacteria, Aquifex, Chlamydia, Spirochaetes, Cytophaga-Chlorobium, and Planctomycetes, are characterized by three distinct inserts, namely a Sandwich Barrel Hybrid Motif domain in the beta subunit, a beta-beta' module (BBM) 1 domain in the beta' subunit, and a distinct helical module in the sigma subunit. The DdRp subunits of remaining bacteria, which comprise Group II, lack these inserts, although some additional inserted domains are present in individual lineages. The separation of bacteria into Group I and Group II is generally compatible with the topologies of phylogenetic trees of the conserved regions of DdRp subunits and concatenated ribosomal proteins and might represent the primary bifurcation in bacterial evolution. A striking deviation from this evolutionary pattern is Aquifex whose DdRp subunits cluster within Group I, whereas phylogenetic analysis of ribosomal proteins identifies Aquifex as grouping with Thermotoga another bacterial hyperthemophile belonging to Group II. The inferred evolutionary scenario for the DdRp subunits includes domain accretion and rearrangement, with some likely horizontal transfer events. Although evolution of bacterial DdRp appeared to be generally dominated by vertical inheritance, horizontal transfer of complete genes for all or some of the subunits, resulting in displacement of the ancestral genes, might have played a role in several lineages, such as Aquifex, Thermotoga, and Fusobacterium.
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PMID:Evolution of bacterial RNA polymerase: implications for large-scale bacterial phylogeny, domain accretion, and horizontal gene transfer. 1519 91

Helicobacter pylori FlgR activates transcription with sigma54-RNA polymerase holoenzyme (sigma54-holoenzyme) from at least five flagellar operons. Activators of sigma54-holoenzyme generally bind enhancer sequences located >70 bp upstream of the promoter and contact sigma54-holoenzyme bound at the promoter through DNA looping to activate transcription. H. pylori FlgR lacks the carboxy-terminal DNA-binding domain present in most sigma54-dependent activators. As little as 42 bp of DNA upstream of the flaB promoter and 26 bp of DNA sequence downstream of the transcriptional start site were sufficient for efficient FlgR-mediated expression from a flaB'-'xylE reporter gene in H. pylori, indicating that FlgR does not use an enhancer to activate transcription. Other examples of sigma54-dependent activators that lack a DNA-binding domain include Chlamydia trachomatis CtcC and activators from the other Chlamydia spp. whose genomes have been sequenced. FlgR from Helicobacter hepaticus and Campylobacter jejuni, which are closely related to H. pylori, appear to have carboxy-terminal DNA-binding domains, suggesting that the loss of the DNA-binding domain from H. pylori FlgR occurred after the divergence of these bacterial species. Removal of the amino-terminal regulatory domain of FlgR resulted in a constitutively active form of the protein that activated transcription from sigma54-dependent genes in Escherichia coli. The truncated FlgR protein also activated transcription with E. coli sigma54-holoenzyme in an in vitro transcription assay.
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PMID:Helicobacter pylori FlgR is an enhancer-independent activator of sigma54-RNA polymerase holoenzyme. 1523 86

All known chlamydiae are either proven human or animal pathogens or possess such potential. Due to increasing reports of chlamydiae diversity in the environment, it is important to develop reliable means for identifying and characterizing Chlamydiales species. The identification of environmental chlamydiae at present relies on their branching pattern in 16S rRNA trees, as well as 16S/23S consensus motifs which display variability. At present, no reliable molecular signatures are known which are unique to all Chlamydiales species. Besides the rRNAs, sequence information for different Chlamydiales is not available for any other gene sequence. In this report, a number of molecular signatures are described that consist of conserved inserts and deletions (indels), in widely distributed proteins [RNA polymerase alpha subunit (RpoA), elongation factor (EF)-Tu, EF-P, DNA gyrase B subunit (GyrB) and lysyl-tRNA synthetase (LysRS)], that are distinctive characteristics of all available chlamydiae homologues (from Chlamydiaeceae species and Parachlamydiae sp. UWE25) and not found in any other bacteria. Using PCR primers for highly conserved regions in these proteins, the corresponding fragments of these genes from Simkania negevensis, Waddlia chondrophila, and in a number of cases for Neochlamydia hartmanellae, covering all families within the phylum Chlamydiae, have been cloned and sequenced. The shared presence of the identified signatures in these species provides strong evidence that these molecular signatures are distinctive characteristics of the entire order Chlamydiales and can be used to reliably determine the presence of chlamydiae or chlamydia-related organisms in environmental samples. The sequence information for these protein fragments was also used to determine the interrelationships among chlamydiae species. In a phylogenetic tree based on a combined dataset of sequences from RpoA, EF-Tu, EF-P and GyrB, the environmental chlamydiae (i.e. Simkania, Waddlia and Parachlamydia) and the traditional Chlamydiaceae (i.e. Chlamydophila and Chlamydia) formed two distinct clades. Similar relationships were also observed in individual protein phylogenies, as well as in a 16S rRNA tree for the same species. These results provide evidence that the divergence between the traditional Chlamydiaceae species and the other chlamydiae families occurred very early in the evolution of this group of bacteria.
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PMID:Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification. 1607 43

Chlamydia species are widely disseminated obligate intracellular pathogens that primarily cause urogenital, ocular, and respiratory infections. In these studies, we show that exposing mammalian cells to antibacterial agents prior to Chlamydia inoculation protects the host cells against subsequent challenge by chlamydiae (the protective effect [PE]). Rifalazil exhibited a considerably stronger PE than did azithromycin, rifampin, doxycycline, and ofloxacin. Specifically, 0.002 microg/ml rifalazil incubated for 1 day with a monolayer of McCoy cells was sufficient to protect against a challenge 2 days later with Chlamydia trachomatis serovar D (UW-3). The PE was observed with five different mammalian cell lines and with a variety of C. trachomatis and Chlamydia pneumoniae isolates. The duration of the PE was 6 to 12 days for rifalazil (depending on the cell line), a maximum of 3 days for azithromycin, and less than a day for the other drugs tested. For rifalazil, the PE was shown to be mediated by inhibition of the chlamydial RNA polymerase since mutants with altered RNA polymerases had correspondingly altered PEs. These results suggest that rifalazil may be unique in its ability to prevent infection with obligate intracellular pathogens for a considerable time after treatment. This characteristic may be of particular public health value in reducing reinfection with chlamydiae.
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PMID:Rifalazil pretreatment of mammalian cell cultures prevents subsequent Chlamydia infection. 1643 94

Tryptophan is an essential amino acid that is required for normal development in Chlamydia species, and tryptophan metabolism has been implicated in chlamydial persistence and tissue tropism. The ability to synthesize tryptophan is not universal among the Chlamydiaceae, but species that have a predicted tryptophan biosynthetic pathway also encode an ortholog of TrpR, a regulator of tryptophan metabolism in many gram-negative bacteria. We show that in Chlamydia trachomatis serovar D, TrpR regulates its own gene and trpB and trpA, the genes for the two subunits of tryptophan synthase. These three genes form an operon that is transcribed by the major form of chlamydial RNA polymerase. TrpR acts as a tryptophan-dependent aporepressor that binds specifically to operator sequences upstream of the trpRBA operon. We also found that TrpR repressed in vitro transcription of trpRBA in a promoter-specific manner, and the level of repression was dependent upon the concentrations of TrpR and tryptophan. Our findings provide a mechanism for chlamydiae to sense changes in tryptophan levels and to respond by modulating expression of the tryptophan biosynthesis genes, and we present a unified model that shows how C. trachomatis can combine transcriptional repression and attenuation to regulate intrachlamydial tryptophan levels. In the face of host defense mechanisms that limit tryptophan availability from the infected cell, the ability to maintain homeostatic control of intrachlamydial tryptophan levels is likely to play an important role in chlamydial pathogenesis.
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PMID:Molecular mechanism of tryptophan-dependent transcriptional regulation in Chlamydia trachomatis. 1674 Sep 30

sigma(28) RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial sigma(28) promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the sigma(28)-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a -35 element recognized by chlamydial sigma(28) RNA polymerase that resembles the consensus -35 sequence. Within the -10 element, however, chlamydial sigma(28) RNA polymerase showed a striking preference for a CGA sequence at positions -12 to -10 rather than the longer consensus -10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli sigma(28) RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the -10 promoter element recognized by sigma(28) RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that sigma(28) RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for sigma(28) RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred sigma(28) promoter that we defined in the context of the hctB promoter is TAAAGwwy-n(11/12)-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n(11/12) is a spacer of 11 or 12 nt.
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PMID:Mutational analysis of the promoter recognized by Chlamydia and Escherichia coli sigma(28) RNA polymerase. 1685 42

The sigma transcription factor confers the promoter recognition specificity of RNA polymerase (RNAP) in eubacteria. Chlamydia trachomatis has three known sigma factors, sigma(66), sigma(54), and sigma(28). We developed two methods to facilitate the characterization of promoter sequences recognized by C. trachomatis sigma(28) (sigma(28)(Ct)). One involved the arabinose-induced expression of plasmid-encoded sigma(28)(Ct) in a strain of Escherichia coli defective in the sigma(28) structural gene, fliA. The second was an analysis of transcription in vitro with a hybrid holoenzyme reconstituted with E. coli RNAP core and recombinant sigma(28)(Ct). These approaches were used to investigate the interactions of sigma(28)(Ct) with the sigma(28)(Ct)-dependent hctB promoter and selected E. coli sigma(28) (sigma(28)(Ec))-dependent promoters, in parallel, compared with the promoter recognition properties of sigma(28)(EC). Our results indicate that RNAP containing sigma(28)(Ct) has at least three characteristics: (i) it is capable of recognizing some but not all sigma(28)(EC)-dependent promoters; (ii) it can distinguish different promoter structures, preferentially activating promoters with upstream AT-rich sequences; and (iii) it possesses a greater flexibility than sigma(28)(EC) in recognizing variants with different spacing lengths separating the -35 and -10 elements of the core promoter.
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PMID:Selective promoter recognition by chlamydial sigma28 holoenzyme. 1693 33


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