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 TyrR protein of Escherichia coli K12 is a homodimer containing 513 amino acids/subunit. This protein is important in the transcriptional regulation of several genes whose protein products catalyze steps in aromatic amino acid biosynthesis or transport. Methods were developed for efficiently purifying the TyrR protein to apparent homogeneity. We analyzed the pattern of cleavage of the TyrR protein by trypsin, either in the absence of ligands or in the presence of saturating levels of L-tyrosine, ATP, or poly(dI-dC). At low (1:200 ratio by weight) trypsin levels, in the absence of ligands, two major digestion products accumulated. These were polypeptides of 22 and 31 kDa, shown to contain amino acid residues 1-190 and 191-467, respectively. The pattern of trypsin cleavage was unaffected by tyrosine. In the presence of ATP, an intermediate species of 53 kDa, probably containing amino acid residues 1-467, was observed. The kinetics of appearance of the 53-kDa species were consistent with a role for ATP in accelerating the hydrolysis of the R467-F468 peptide bond. The 53-kDa polypeptide underwent further tryptic hydrolysis to yield fragments of 22 and 31 kDa. When both tyrosine and ATP were present, the rate of formation of the 22- and 31-kDa fragments was more rapid than in the absence of these ligands. It appears that when both ligands are bound, the rates of hydrolysis of peptide bonds R190-Q191 and R467-F468 are both enhanced. Additional limited proteolysis experiments suggested that polypeptide segment 191-467 contains ATP binding site(s), and that the rate of cleavage of peptide bonds R190-Q191 and R467-F468 is altered when the TyrR protein interacts with poly(dI-dC), an analog of target DNA. Our results reveal the presence of two major structural domains within the TyrR protein. The first domain (amino acid residues 1-190) is extremely resistant to hydrolysis by trypsin. The second domain (residues 191-467), which is likely to contain ATP-binding site(s), is homologous to several other transcriptional activators specific for promoters responsive to the sigma 54 form of RNA polymerase. The remainder of the TyrR protein (residues 468-513) contains the operator recognition elements, probably arranged in the form of a helix-turn-helix motif. This polypeptide segment was not detected as a discrete tryptic hydrolysis product.
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PMID:The TyrR protein of Escherichia coli, analysis by limited proteolysis of domain structure and ligand-mediated conformational changes. 844 80

Activated human and rat T cells as well as mouse T-cell clones have been reported to synthesize and express major histocompatibility complex (MHC) class II molecules. However, the capacity of class II+ antigen (Ag) presenting T cells to induce proliferation of Ag-specific cloned T cells has been controversial. We analysed whether the failure of some T-cell clones to proliferate in response to Ag presented by class II+ T cells is because of a lack of costimulatory cytokine production by the antigen-presenting cells (APC). As a model system the mouse class II+ cloned BI/O4.1 T cells were used as APC in order to activate the T cell clone KIII5. This T-helper 1 (Th1) type, GAT (synthetic copolymer of L-glutamic acid, L-alanine and L-tyrosine)-specific clone is characterized by an efficient downregulation of interleukin-2 receptor (IL-2R) with time following antigenic stimulation. KIII5 cells respond to GAT-presenting splenic antigen-presenting cells (APC) by IL-2 production, IL-2R upregulation and proliferation. When BI/O4.1 T cells were used as APC, KIII5 cells produced IL-2, but did not proliferate. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed a lack of IL-12 production by BI/O4.1 cells. Addition of IL-12 to a coculture of Ag-presenting BI/O4.1 cells and KIII5 cells fully reconstituted a proliferative response. IL-12 in synergy with IL-2 upregulated IL-2R alpha chain expression and enhanced proliferation of KIII5 cells. Our data suggest, that class II+ T cells are not functional in inducing Ag-mediated expansion of resting Th1 cells owing to their failure to produce IL-12, but rather that they play a role in amplification loops during an ongoing immune response.
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PMID:Costimulatory signalling potential of murine MHC class II-positive T-clone cells. 895 51

Osmotic regulation of proU expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the sigma70-controlled promoter of proU in Escherichia coli, we were able to demonstrate a superposed TyrR-mediated activation by L-phenylalanine (Phe), as well as repression by L-tyrosine, of proU expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of proU at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in E. coli.
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PMID:Superimposition of tyrR protein-mediated regulation on osmoresponsive transcription of Escherichia coli proU in vivo. 985 23

Pseudomonas putida uses L-phenylalanine as the sole nitrogen source for growth by converting L-phenylalanine to L-tyrosine, which acts as a donor of the amino group. This metabolic step requires the products of the phhA and phhB genes, which form an operon. Expression of the phhA promoter is mediated by the phhR gene product in the presence of L-phenylalanine or L-tyrosine. The PhhR protein belongs to the NtrC family of enhancers. In contrast with most members of this family of regulators, transcription from the promoter of the phhAB operon (P(phhA)) is mediated by RNA polymerase with sigma(70) rather than with sigma(54). The PhhR regulator binds two similar but non-identical upstream PhhR motifs (5'-TGTAAAATTATCGTTACG-3' and 5'-ACAAAAACTGTGTTTCCG-3') that are located 39 and 97 nucleotides upstream of the proposed -35 hexamer for RNA polymerase, respectively. These motifs are called PhhR proximal and PhhR distal binding motifs because of their position with respect to the RNA polymerase binding site. Affinity of PhhR for its target sequences was determined by isothermal titration calorimetry and was found to be around 30 nM for the proximal site and 2 microM for the distal site, and the binding stoichiometry is of a dimer per binding site. Both target sequences are sine qua non requirements for transcription, since inactivation of either of them resulted in no transcription from the phhA promoter. An IHF binding site overlaps the proximal PhhR proximal motif, which is recognized by IHF with a K(D) of around 1.2 microM. IHF may consequently compete with PhhR for binding and indeed inhibits PhhR-dependent phhAB operon expression.
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PMID:Catabolism of phenylalanine by Pseudomonas putida: the NtrC-family PhhR regulator binds to two sites upstream from the phhA gene and stimulates transcription with sigma70. 1721 60

Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, L-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.
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PMID:Mutagenesis of the bacterial RNA polymerase alpha subunit for improvement of complex phenotypes. 1925 86

The phhAB operon encodes a phenylalanine hydroxylase involved in the conversion of L-phenylalanine into L-tyrosine in Pseudomonas putida. The phhAB promoter is transcribed by RNA polymerase sigma-70 and is unusual in that the specific regulator PhhR acts as an enhancer protein that binds to two distant upstream sites (-75 to -92 and -132 to -149). There is an integration host factor (IHF) binding site that overlaps the proximal PhhR box, and, consequently, IHF acts as an inhibitor of transcription. Use of L-phenylalanine is compromised in a crp-deficient background due to reduced expression from the phhAB promoter. Electrophoretic mobility shift assays and DNase I footprinting assays reveal that Crp binds at a site centered at -109 only in the presence of cyclic AMP (cAMP). We show, using circular permutation analysis, that the simultaneous binding of Crp/cAMP and PhhR bends DNA to bring positive regulators and RNA polymerase into close proximity. This nucleoprotein complex promotes transcription from phhA only in response to L-phenylalanine.
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PMID:Involvement of the global Crp regulator in cyclic AMP-dependent utilization of aromatic amino acids by Pseudomonas putida. 2208 86