Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity. Purified T. maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity. The complexes formed between nucleic acid and T. maritima NusG were unable to penetrate a polyacrylamide or agarose gel. The affinity of the protein for DNA was highest in buffers containing about 50 mM salt. The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T. maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA. The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA. Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis. Neither the wild-type T. maritima nusG gene nor a deletion derivative more similar to the E. coli gene was able to substitute for the essential E. coli nusG. Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T. maritima NusG, and the other has only the sequences present in NusG homologs from E. coli and other eubacteria. Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T. maritima NusG.
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PMID:A NusG-like protein from Thermotoga maritima binds to DNA and RNA. 876 36

On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a -35 hexamer region of -42 aaaaaga TATACAaa -28 and an unusual, GC-rich -10 hexamer region of -13 tTATCGCt -6. The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations. The 5' extent of P2 was located at bp -42. Replacement of wild-type sequence with two G's at positions -41 and 40, -35 and 34, and -29 and 28 resulted in severely decreased transcription. Additionally, the spacing between the -35 and -10 hexamers could not be shortened without adversely affecting in vitro activity. Substitution of G at position -13, -10, -7, or -6 had little or no effect on transcription, whereas substitution of G at -11 or -12 significantly decreased promoter strength. Triple point mutations which changed the -10 hexamer from TATCGC to TATTAT,TATATT, or TATAAT had little effect on promoter activity. Unlike the partially purified C. trachomatis sigma66-RNA polymerase used in this study, purified Escherichia coli sigma70-RNA polymerase did not recognize the wild-type P2 promoter. Mutant P2 templates with -10 hexamers that resembled the consensus recognition site were transcribed by E. coli holoenzyme in vitro, suggesting that C. trachomatis sigma66-RNA polymerase has special promoter recognition properties not found in E. coli sigma70-holoenzyme.
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PMID:Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis. 882 99

The past decade has seen the rapid advancement of molecular biology and its application in the field of infectious diseases. The polymerase chain reaction (PCR) is a technique which brings about the in vitro amplification of DNA, and is clinically useful in the sensitive, specific and rapid diagnosis of various infectious diseases. The fast diagnosis of viral infections using PCR is a prime example, since viral culture may take weeks to grow and since serologic conversion seldom occurs until the convalescent phase of the clinical illness. Similarly, PCR is applicable to the diagnosis of pulmonary infections caused by mycobacteria, Legionella, methicillin-resistant Staphylococcus aureus, anaerobes, Mycoplasma, Chlamydia, Pneumocystis carinii and so on. On the other hand, the drug-resistant phenotype of a microorganism is predictable by the detection of gene alterations related to the drug resistance. For example, the deletion of the catalase-peroxidase gene relating to isoniazid resistance of mycobacteria, and point mutations in the RNA polymerase beta subunit (rpoB) gene relating to rifampicin resistance have been elucidated. As these applied techniques become more widely available and less costly, they should contribute not only to the rapid diagnosis of infectious diseases, but also to the adequate selection of antibiotics and the shortening of hospitalization.
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PMID:[Advances in genetic diagnostics for respiratory tract infections]. 883 14

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.
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PMID:Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression. 895 11

Chlamydia trachomatis RNA polymerase was partially purified by heparin-agarose chromatography and used in conjunction with a plasmid-borne G-less cassette template to characterize the C. trachomatis rRNA P1 promoter in vitro. Stepwise mutational analysis revealed that sequences in the -10, -25, and -35 regions are necessary for promoter activity, but no sequence upstream of position -40 is required. Partially purified C. trachomatis RNA polymerase and purified Escherichia coli holoenzyme exhibited some differences in promoter specificity.
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PMID:Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter. 895 22

The transcriptional organization and regulation of the dnaK and groE heat shock operons of Chlamydia trachomatis were studied and found to resemble those of the cognate operons of Bacillus subtilis and Clostridium acetobutylicum. The gene order is conserved (hrcA-grpE-dnaK), but no dnaJ homolog could be identified in this region. The dnaK operon was transcribed as a low-abundance polycistronic mRNA whose levels did not increase upon exposure to heat shock. In contrast, a more abundant 2.3-kb mRNA encoding only the dnaK sequence was detectable, and its steady-state level increased upon heat shock. The transcription initiation sites of the dnaK and groE operons were found to be preceded by sequences that resemble an Escherichia coli sigma70 consensus promoter. Upstream of each putative promoter is an inverted repeat sequence which resembles a similar element (CIRCE [controlling inverted repeat of chaperone expression]) found upstream of the dnaK and groE operons in at least 27 eubacterial species. In vitro transcription studies utilizing partially purified C. trachomatis RNA polymerase demonstrated that the regions containing the putative promoter elements of the dnaK and groE operons are functional, although heat shock-regulated expression could not be demonstrated.
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PMID:Transcriptional organization and regulation of the dnaK and groE operons of Chlamydia trachomatis. 895 23

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.
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PMID:The chlamydial EUO gene encodes a histone H1-specific protease. 929 54

We have characterized the Chlamydia trachomatis ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified C. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential -10 and -35 elements, analogous to Escherichia coli promoters recognized by E-sigma70. We identified a novel AT-rich region immediately downstream of the -35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the -35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.
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PMID:Mutational analysis of the Chlamydia trachomatis rRNA P1 promoter defines four regions important for transcription in vitro. 957 86

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole, termed an inclusion. To identify chlamydial proteins that are unique to the intracellular phase of the life cycle, a lambda expression library of Chlamydia psittaci DNA was differentially screened with convalescent antisera from infected guinea pigs and antisera directed at formalin-fixed purified chlamydial elementary bodies (EBs). One library clone was identified that harboured two open reading frames (ORFs) with coding potential for similar-sized proteins of approximately 20 kDa. These proteins were subsequently termed IncB and IncC. Sequencing of the cloned insert revealed a strong Escherichia coli-like promoter sequence immediately upstream of incB and a 36nt intergenic region between the ORFs. Sequence analysis of the region upstream of incB and incC revealed two ORFs that had strong homologies to an amino acid transporter and a sodium-dependent transporter. Immunoblotting with antisera directed at IncB or IncC demonstrated that these proteins are present in C. psittaci-infected HeLa cells but are absent or below the level of detection in purified EBs. Reverse transcriptase-polymerase chain reactions provided evidence that incB and incC are transcribed in an operon. Immunofluorescence microscopy demonstrated that IncB and IncC are each localized to the inclusion membrane of infected cells. No primary sequence similarity is evident between IncA, IncB or IncC, but each contains a large hydrophobic domain of similar size and character as in IncA. Analysis of the recently completed C. trachomatis serovar D genome database has revealed C. trachomatis ORFs encoding homologues to incB and incC, indicating that these genes are conserved among the chlamydiae.
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PMID:Tandem genes of Chlamydia psittaci that encode proteins localized to the inclusion membrane. 966 87

L7 is one of the ribosomal proteins frequently targeted by autoantibodies in rheumatic autoimmune diseases. A computer search revealed a region within the immunodominant epitope of L7 (peptide II) that is highly homologous to amino acid sequence 264-286 of the RNA polymerase major sigma factor of the eubacterium Chlamydia trachomatis. Anti-L7 autoantibodies affinity purified from the immunodominant epitope were able to recognize this sequence as they reacted with purified recombinant sigma factor. Immunofluorescence labeling experiments on C. trachomatis lysates revealed a punctate staining pattern of numerous spots when incubated with the affinity-purified anti-peptide II autoantibodies. Binding of autoantibodies to peptide II was inhibited by the homologous sigma peptide. This is the first demonstration of epitope mimicry between a human and a chlamydial protein on the level of B cells. Antibody screening revealed a significant correlation between the presence of anti-L7 autoantibodies and C. trachomatis infection in patients with systemic lupus erythematosus and mixed connective tissue disease. Our results suggest that molecular mimicry is involved in the initiation of anti-L7 autoantibody response and may represent a first glance into the immunopathology of Chlamydia with respect to systemic rheumatic diseases.
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PMID:Correlation between chlamydial infection and autoimmune response: molecular mimicry between RNA polymerase major sigma subunit from Chlamydia trachomatis and human L7. 984 29


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