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)

RNA transcribed in vitro from DNA of a tryptophan (trp) transducing strain of bacteriophage phi80 which contains the trp regulatory elements consists of a polycistronic messenger transcribed from the structural genes, and possibly the regulatory region, and a separate RNA species (called trp regRNA) which is transcribed from the regulatory region. This conclusion is based on hybridization experiments with trp RNA synthesized in vitro and the separate DNA strands of trp transducing strains of lambda with and without the trp regulatory elements. The length of trp regRNA determined by filtration on Sephadex G-200 is 110-180 nucleotides. From the amount and the length of trp regRNA we have calculated that 8-20 copies of trp regRNA are synthesized per copy of polycistronic trp mRNA. We conclude that during transcription of the trp operon RNA polymerase frequently is rejected at a specific site ahead of the first structural gene, trpE. The termination factor Rho is not involved in this process. A different protein fraction, which specifically stimulates the synthesis of trp enzymes in an in vitro protein-synthesizing system (Pouwels and Van Rotterdam, 1975), was found to antagonize the abortive synthesis of trp mRNA. A model is proposed for the control of transcription of the trp genes, which operates through a mechanism of punctuation of RNA synthesis at a specific site on the DNA template and anti-termination of RNA synthesis by means of a positive control factor.
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PMID:Punctuation of transcription in vitro of the tryptophan operon of Escherichia coli. A novel type of control of transcription. 1609 71

A protein fraction, called At (= anti termination) factor, has been isolated from extracts of E. coli and partially purified. The At factor stimulates the synthesis in vitro of anthranilate synthetase, an enzyme encoded by two genes of the tryptophan (trp) operon, but has no effect on the synthesis of T7 RNA polymerase and other T7- and T4 coded proteins. The At factor stimulates the synthesis of trp mRNA; it has no effect on the translation of trp mRNA. We conclude that in vitro transcription of the trp operon is positively controlled.
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PMID:In vitro synthesis of enzymes of the tryptophan operon of Escherichia coli. Evidence for positive control of transcription. 1609 72

Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNA(Trp) available to translate the second codon of the trpY mRNA.
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PMID:Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus. 1615 76

Lack of maturation of phagosomes containing pathogenic Mycobacterium tuberculosis within macrophages has been widely recognized as a crucial factor for the persistence of mycobacterial pathogen. Host molecule tryptophan-aspartate containing coat protein (TACO) has been shown to play a crucial role in the arrest of such a maturation process. The present study was addressed to understand whether or not polyphenols derived from green tea could down-regulate TACO gene transcription. And if yes, what impact TACO gene down-regulation has on the uptake/survival of M. tuberculosis within macrophages. The reverse-transcriptase polymerase chain reaction and reporter assay technology, employed in this study, revealed that the major component of green tea polyphenols, epigallocatechin-3-gallate had the inherent capacity to down-regulate TACO gene transcription within human macrophages through its ability to inhibit Sp1 transcription factor. We also found out that TACO gene promoter does contain Sp1 binding sequence using bioinformatics tools. The down-regulation of TACO gene expression by epigallocatechin-3-gallate was accompanied by inhibition of mycobacterium survival within macrophages as assessed through flow cytometry and colony counts. Based on these results, we propose that epigallocatechin-3-gallate may be of importance in the prevention of tuberculosis infection.
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PMID:Green tea polyphenol inhibits Mycobacterium tuberculosis survival within human macrophages. 1635 57

Arom gene, encoding a single polypeptide that catalyses steps two to six of the aromatic amino acid (phenylalanine, tyrosine and tryptophan) biosynthetic pathway, has been amplified from Scleortinia sclerotiorum genomic DNA by PCR and sequenced. In order to identify the fragment encoding AROM protein experimentally and search a method of obtaining the enzyme in a large amount, the open reading frame of arom gene of S. sclerotiorum was amplified by Pyrobest DNA Polymerase and inserted between Kpn I and Not I sites of the vector pYES2 to construct the expression vector pYES2-arom. The construct was transformed into Saccharomyces cerevisiae H158 by the method of LiAc/ SSDNA/PEG. The rate of transformation was 2 x 10(2)/microg DNA, which was enough for the selection of the positive transformants. PCR using the extracted plasmids as the templates and restriction enzyme analysis of the plasmids extracted from E. coli cells transformed by the above plasmids were performed respectively to screen the positive S. cerevisiae transformants since the copy number of the plasmid in S. cerevisiae was low. Subsequently, the transformant activated by the SC-U medium containing 2% raffinose was inoculated into the SC-U medium containing 2% galactose and the SC-U medium containing 2% glucose respectively to induce and depress the expression of the foreign arom gene. The results of RT-PCR analysis showed: there was not any DNA band in the negative control without the anti-transcriptase, which indicated there was no DNA contamination in the extracted total RNA; there was an expected DNA band in the positive control using the expression vector pYES2-arom as the template, which indicated the used amplification condition was proper; there was not any DNA band in the negative control using total RNA from the depressed transformant as the template, which indicated the DNA bands amplified from total RNA of the induced transformant were not false; there were the expected DNA bands in the samples using total RNA of the transformant induced for 48h, 60h, 72h or 84h as the templates, which indicated the heterogeneous arom gene was transcribed in S. cerevisiae H158 cells. The result of Northern hybridization was consistent with that of RT-PCR, and showed that arom gene of S. sclerotiorum had been transcribed in S. cerevisiae H158 cells when the cells were induced for 48h in the SC-U medium containing 2% galactose at 30 degrees C at 180r/min. 5-enolpyruvylshikimate-3-phosphate synthase activity of the transformant, which was one of AROM protein activities, was measured by estimating the rate of Pi release to check the expressed AROM protein was active or not. The results of enzyme assay in the different culture period indicated that the transformant had 5-enolpyruvylshikimate-3-phosphate synthase activity and the activity reached the peak when the transformant was induced for 72h in SC-U medium at 30 degrees C at 180r/min. The molecular weight of AROM protein is high, it exists in cytoplasm as a dimmer and its expression is controlled by the amounts of amino acids. Therefore, it is very difficult to purify the enzyme. A great lot protein can be obtained by heterogeneous expression. S. cerevisiae expression system has the merits of safe status, authentic posttranslational modification, fast cultivation etc. and usually is the first choice eukaryotic expression system. S. cerevisiae expression system of AROM protein from S. sclerotiorum was successfully constructed for the first time, which provided the basis for the research on the catalysis mechanism of the enzyme and an economical means of simultaneously synthesizing five aromatic amino acid biosynthetic pathway enzymes.
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PMID:[Cloning and expression of arom gene of Sclerotinia sclerotiorum]. 1657 63

We have isolated, sequenced, and expressed a cold-specific cDNA clone, Wcs120, that specifically hybridizes to a major mRNA species of approximately 1650 nucleotides from cold-acclimated wheat (Triticum aestivum L.). The accumulation of this mRNA was induced in less than 24 hours of cold treatment, and remained at a high steady-state level during the entire period of cold acclimation in the two freezing-tolerant genotypes of wheat tested. The expression of Wcs120 was transient in a less-tolerant genotype even though the genomic organization of the Wcs120 and the relative copy number were the same in the three genotypes. The mRNA level decreased rapidly during deacclimation and was not induced by heat shock, drought, or abscisic acid. The Wcs120 cDNA contains a long open reading frame encoding a protein of 390 amino acids. The encoded protein is boiling stable, highly hydrophilic, and has a compositional bias for glycine (26.7%), threonine (16.7%), and histidine (10.8%), although cysteine, phenylalanine, and tryptophan were absent. The WCS120 protein contains two repeated domains. Domain A has the consensus amino acid sequence GEKKGVMENIKEKLPGGHGDHQQ, which is repeated 6 times, whereas domain B has the sequence TGGTYGQQGHTGTT, which is repeated 11 times. The two domains were also found in barley dehydrins and rice abscisic acid-induced protein families. The expression of this cDNA in Escherichia coli, using the T(7) RNA polymerase promoter, produced a protein of 50 kilodaltons with an isoelectric point of 7.3, and this product comigrated with a major protein synthesized in vivo and in vitro during cold acclimation.
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PMID:Cloning, characterization, and expression of a cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat. 1666 48

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

In this article a model, first, classical attenuation RNA regulation of gene expression by means of transcription termination is offered. The model bases on representation about a macrostate of secondary structure in RNA regulatory region between a ribosome and a RNA polymerase, on the formulas of a resonant type defining the value of deceleration of a RNA polymerase by a set of hairpins in the same region. The special attention is given to selection of parameters of model. To check of model the computer simulation is carried out and the dependences of transcription termination probability from the value of concentration charged tRNA are obtained, in particular, and from concentration of amino acid for many regulatory regions in genomes of bacteria (here data are presented for trpE genes in Streptomyces spp., Bradyrhizobium japonicum and Escherichia coli) and at various values of three parameters, which authors consider as the main. The obtained dependences are compounded with the accessible experimental data; including, under the form of the graphs concerning to activity of an enzyme depending on concentration of amino acid (for example, anthranilate synthase from tryptophan in S. venezuela). One possible usage: now attenuation is predicted usually by means of multiple alignment, it needs some sequences; the obtaining with the help of model on an individual sequence characteristic for attenuation or its absence of a curve at approaching parameters could be considered as argument for the benefit of presence or absence of attenuation.
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PMID:[Model of genes expression regulation in bacteria by means of formation of secondary RNA structures]. 1681 69

sigma factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) sigma factors govern the environment dependent regulation of transcription. ECF sigma factors have two domains sigma(2) and sigma(4) that recognize the -10 and -35 promoter elements. However, unlike the primary sigma factor sigma(A), the ECF sigma factors lack sigma(3), a region that helps in the recognition of the extended -10 element and sigma(1.1), a domain involved in the autoinhibition of sigma(A) in the absence of core RNA polymerase. Mycobacterium tuberculosis sigma(C) is an ECF sigma factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF sigma factors, sigma(C) does not appear to have a regulatory anti-sigma factor located in the same operon. We also note that M. tuberculosis sigma(C) differs from the canonical ECF sigma factors as it has an N-terminal domain comprising of 126 amino acids that precedes the sigma(C)(2) and sigma(C)(4) domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the sigma(C)(2) and sigma(C)(4) domains of sigma(C) were determined. These promoter recognition domains are structurally similar to the corresponding domains of sigma(A) despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of sigma(C)(2) as well as surface plasmon resonance measurements reveal that the sigma(C)(2) and sigma(C)(4) domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of sigma(C)(2) is involved in this interdomain interaction. Interaction between the promoter recognition domains in M. tuberculosis sigma(C) are thus likely to regulate the activity of this protein even in the absence of an anti-sigma factor.
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PMID:Structural and biophysical studies on two promoter recognition domains of the extra-cytoplasmic function sigma factor sigma(C) from Mycobacterium tuberculosis. 1714 60

The RpoS subunit of RNA polymerase controls the expression of numerous genes involved in stationary phase and in response to different stress conditions. The regulatory protein Crl increases the activity of RpoS by direct interaction with the RpoS holoenzyme. To define the extent of the Crl regulon, we used two-dimensional SDS-PAGE to measure the role of Crl in regulating the expression of the Escherichia coliproteome in stationary phase at 30 degrees C. By comparing the proteome of four strains (wild type, crl(-), rpoS(-), and crl(-)rpoS(-)), we observed that the intensity of 74 spots was modified in at least one mutant context. 62 spots were identified by mass spectrometry and correspond to 40 distinct proteins. They were classified in four main categories: DNA metabolism, central metabolism, response to environmental modifications, and miscellaneous. Three proteins were specifically involved in quorum sensing: TnaA (the tryptophanase that converts tryptophan to indole), WrbA (Trp repressor-binding protein), and YgaG (homologous to LuxS, autoinducer-2 synthase). Because little is known about the regulation of Crl expression, we investigated the influence of diffusible molecules on the expression of Crl. Using Western blotting experiments, we showed that, at 30 degrees C, a diffusible molecule(s) produced during the transition phase between the exponential and stationary phases induces a premature expression of Crl. Indole was tested as one of the potential candidates: at 37 degrees C, it is present in the extracellular medium at a constant concentration, but at 30 degrees C, its concentration peaks during the transition phase. When indole was added to the culture medium, it also induced prematurely the expression of Crl at both the transcriptional and translational levels in a Crl-dependent manner. Crl may thus be considered a new environmental sensor via the indole concentration.
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PMID:The Crl-RpoS regulon of Escherichia coli. 1722 7


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