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 trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms. Both mechanisms require the binding of tryptophan-activated TRAP to the 11 (G/U)AG-repeat segment in the trp leader transcript. To promote termination, TRAP must bind to the nascent RNA before the antiterminator structure forms. Because only 20 nucleotides separate the TRAP-binding site from the 3' end of the antiterminator, TRAP has a short time frame to control this regulatory decision. Synchronization of factor binding and/or RNA folding with the RNA polymerase position is a major challenge in all attenuation mechanisms. Because RNA polymerase pausing allows this synchronization in many attenuation mechanisms, we performed experiments in vitro to determine whether pausing participates in the B. subtilis trp attenuation mechanism. We identified two NusA-stimulated pause sites in the trp leader region. Formation of pause hairpins participates in pausing at both positions. The first pause occurred at the nucleotide just preceding the critical overlap between the alternative antiterminator and terminator structures. TRAP binding to transcripts containing preexisting pause complexes releases RNA polymerase, suggesting that pausing provides additional time for TRAP to bind and promote termination. The second pause is downstream from the trp leader termination point, raising the possibility that this pause event participates in the trpE translation control mechanism. NusA also increases the efficiency of termination in the trp leader region and shifts termination one nucleotide upstream. Finally, NusA-stimulated termination is cooperative, suggesting that binding of multiple NusA molecules influences termination.
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PMID:NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism invitro. 1216 62

Omega (omega), consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of omega in which it helps to maintain beta' in a correct conformation and recruit it to the alpha(2)beta subassembly to form a functional core enzyme (alpha(2)betabeta'omega). The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch onto the N-terminal and C-terminal ends of the beta'-subunit. In the present study we have monitored the conformational changes in beta' as the denatured protein is refolded in the presence and absence of omega using tryptophan fluorescence emission of beta' as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of omega is helpful in beta' refolding. We have also monitored the behavior of the C-terminal tail of omega by engineering three cysteine residues at three different sites in omega and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of omega is mobile in the free protein and gets restrained in the presence of beta'. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the beta' subunit whereas the end of the C-terminal of omega, although it is restrained in the presence of beta', has no interacting partner within a 4-A radius.
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PMID:Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with omega-beta' interaction. 1272 85

RNA helicase A (RHA) is a member of ATPase/helicase and regulates the transcription through recruitment of Pol II and/or by ATP dependent mechanisms. In CREB-dependent transcription, RHA recruits RNA polymerase (Pol) II to the CREB binding protein (CBP) via the minimal transactivation domain (MTAD). This region is well conserved among RHA homologues, whereas it is unique to RHA. The three conserved tryptophan residues in MTAD are critical for transactivation. To understand the importance of tryptophan residues on transactivation, we generated mutants in which tryptophan residues were replaced by other aromatic, bulky hydrophobic or small hydrophobic amino acids. Substitutions of tryptophan with either bulky hydrophobic or small hydrophobic amino acid decreased transcriptional activity, whereas aromatic residue had no effect. Moreover, these mutants with tryptophan to phenylalanine, activated CREB-dependent transcription. These results indicate that aromatic characteristics of tryptophan residues in MTAD are important for CREB-dependent transcription via RHA.
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PMID:Aromatic residues are required for RNA helicase A mediated transactivation. 1285 13

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.
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PMID:Kinetics of transcription initiation at lacP1. Multiple roles of cyclic AMP receptor protein. 1288 19

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan catabolic enzyme that is widely distributed in various tissues. In peripheral blood mononuclear cells (PBMCs), production of IDO by macrophages or dendritic cells has been reported to inhibit T-cell activation and proliferation. In the present study, we have determined that other phenotypes of PBMCs also express IDO. In cultures of PBMCs, IDO was found predominantly in monocyte by immunohistochemistry. Reverse transcriptase polymerase chain reaction analysis showed that IDO mRNA was expressed in T lymphocytes, B lymphocytes and natural killer (NK) cells and that expression was increased upon activation with interferon-gamma. The cytotoxicity of NK cells against K562 and HepG2 cells was reduced by IDO inhibitor. These results suggest that IDO in NK cells is essential for NK cells to generate killing activity against cancer cells.
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PMID:Indoleamine 2,3-dioxygenase is necessary for cytolytic activity of natural killer cells. 1487 Dec 94

The tyrP gene of Escherichia coli encodes a tyrosine specific transporter. Its synthesis is repressed by tyrosine but is activated by phenylalanine and to a lesser extent by tryptophan. Both of these effects are mediated by the TyrR protein when it binds to one or both of its cognate binding sites (TyrR boxes) which encompass nucleotides -30 to -75. Activation in the presence of phenylalanine or tryptophan involves a dimer binding to the upstream box and interacting with the alpha subunit (alphaCTD) of RNA polymerase (RNAP). Repression in the presence of tyrosine involves a hexamer binding to both TyrR boxes. The molecular basis for this repression has been studied in vitro. Whereas initial gel shift experiments fail to show the exclusion of RNAP from the promoter region when TyrR hexamer is bound, a DNase I analysis of slices from the gel shows that in the presence of TyrR, RNAP now binds to a previously unrecognized upstream promoter. Although this upstream promoter is bound strongly by RNAP and forms an open complex on linear DNA templates, it fails to form an open complex on supercoiled templates in vitro and is unable to initiate transcription in vivo. A subsequent gel shift assay using a tyrP fragment which eliminates the upstream RNAP binding site confirms conclusively that, in the presence of tyrosine and ATP, the TyrR protein prevents RNAP from binding to the tyrP promoter. In vitro studies have also been carried out in the presence of TyrR protein and phenylalanine. Binding of TyrR protein to the upstream TyrR box in the presence of phenylalanine is shown to increase the affinity of RNAP for the promoter and stimulate open complex formation at the -10 region of the tyrP promoter. This observation coupled with the results from mutational analysis supports the proposal that TyrR-phenylalanine activates tyrP transcription by stimulating the onset of open complex formation.
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PMID:Mode of action of the TyrR protein: repression and activation of the tyrP promoter of Escherichia coli. 1504 24

The TyrR protein of Escherichia coli can act both as a repressor and as an activator of transcription. It can interact with each of the three aromatic amino acids, with ATP and, under certain circumstances, with the C-terminal region of the alpha-subunit of RNA polymerase. TyrR protein is a dimer in solution but in the presence of tyrosine and ATP it self-associates to form a hexamer. Whereas TyrR dimers can, in the absence of any aromatic amino acids, bind to certain recognition sequences referred to as 'strong TyrR boxes', hexamers can bind to extended sequences including lower-affinity sites called 'weak TyrR boxes', some of which overlap the promoter. There is no single mechanism for repression, which in some cases involves exclusion of RNA polymerase from the promoter and in others, interference with the ability of bound RNA polymerase to form open complexes or to exit the promoter. When bound to a site upstream of certain promoters, TyrR protein in the presence of phenylalanine, tyrosine or tryptophan can interact with the alpha-subunit of RNA polymerase to activate transcription. In one unusual case, activation of a non-productive promoter is used to repress transcription from a promoter on the opposite strand. Regulation of individual transcription units within the regulon reflects their physiological function and is determined by the position and nature of the recognition sites (TyrR boxes) associated with each of the promoters. The intracellular levels of the various forms of the TyrR protein are also postulated to be of critical importance in determining regulatory outcomes. TyrR protein remains a paradigm for a regulator that is able to interact with multiple cofactors and exert a range of regulatory effects by forming different oligomers on DNA and making contact with other proteins. A recent analysis identifying putative TyrR boxes in the E. coli genome raises the possibility that the TyrR regulon may extend beyond the well-characterized transcription units described in this review.
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PMID:The TyrR regulon. 1561 13

Analysis of the genome sequence of the small hyperthermophilic archaeal parasite Nanoarchaeum equitans has not revealed genes encoding the glutamate, histidine, tryptophan and initiator methionine transfer RNA species. Here we develop a computational approach to genome analysis that searches for widely separated genes encoding tRNA halves that, on the basis of structural prediction, could form intact tRNA molecules. A search of the N. equitans genome reveals nine genes that encode tRNA halves; together they account for the missing tRNA genes. The tRNA sequences are split after the anticodon-adjacent position 37, the normal location of tRNA introns. The terminal sequences can be accommodated in an intervening sequence that includes a 12-14-nucleotide GC-rich RNA duplex between the end of the 5' tRNA half and the beginning of the 3' tRNA half. Reverse transcriptase polymerase chain reaction and aminoacylation experiments of N. equitans tRNA demonstrated maturation to full-size tRNA and acceptor activity of the tRNA(His) and tRNA(Glu) species predicted in silico. As the joining mechanism possibly involves tRNA trans-splicing, the presence of an intron might have been required for early tRNA synthesis.
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PMID:Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5'- and 3'-halves. 1569 44

The presence of albumin in the human epidermis has been reported more than a decade ago, but until now, it was assumed that this protein is synthesized in the liver and transported to the avascular skin. To our knowledge, transcription of albumin in the human epidermis was never considered. In this report, we present for the first time evidence for autocrine synthesis of albumin in the human epidermis in keratinocytes in situ and in vitro. Using double immunofluorescence labelling, we identified that albumin colocalized together with its transcription factor PCD/DCoH/HNF-1alpha in suprabasal keratinocytes in human full-thickness skin sections and in keratinocytes cultured in serum-free medium. Moreover, albumin and HNF-1alpha protein expression was confirmed by Western blotting in undifferentiated and differentiated keratinocytes as well as in human epidermal suction blister roof extracts. Reverse-transcriptase polymerase chain reaction analysis from human epidermal keratinocytes and epidermal suction blister roofs revealed the transcription of albumin. Using in vivo fluorescence excitation spectroscopy at the surface of human skin, we confirmed albumin as a major constituent yielding a lambda(max) at 295 nm, which was assigned to the single tryptophan 214 fluorophore in this protein. This in vivo result is in agreement with albumin concentrations of 10(-3) M, underlining the importance of this protein in epidermal homeostasis.
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PMID:In vivo and in vitro evidence for autocrine DCoH/HNF-1alpha transcription of albumin in the human epidermis. 1574 May 90

Rice (Oryza sativa) anthranilate synthase alpha-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.
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PMID:Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway. 1604 Jun 54


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