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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription of the proP gene, encoding a transporter of the osmoprotectants proline and glycine betaine, is controlled from two promoters, P1 and P2, that respond primarily to osmotic and stationary-phase signals, respectively. The P1 promoter is normally expressed at a very low level under low or normal medium osmolarity. We demonstrate that the binding of the cyclic AMP (cAMP) receptor protein (CRP) to a site centered at -34.5 within the promoter is responsible for the low promoter activity under these conditions. A brief period of reduced CRP binding in early log phase corresponds to a transient burst of P1 transcription upon resumption of growth in Luria-Bertani broth. A CRP binding-site mutation or the absence of a functional crp gene leads to high constitutive expression of P1. We show that the binding of CRP-cAMP inhibits transcription by purified RNA polymerase in vitro at P1, but this repression is relieved at moderately high potassium glutamate concentrations. Likewise, open-complex formation at P1 in vivo is inhibited by the presence of CRP under low-osmolarity conditions. Because P1 expression can be further induced by osmotic upshifts in a delta crp strain or in the presence of the CRP binding-site mutation, additional controls exist to osmotically regulate P1 expression.
...
PMID:Cyclic AMP receptor protein functions as a repressor of the osmotically inducible promoter proP P1 in Escherichia coli. 907 29

The proU operon in enterobacteria encodes a binding-protein-dependent transporter for the active uptake of glycine betaine and L-proline, and serves an adaptive role during growth of cells in hyperosmolar environments. Transcription of proU is induced 400-fold under these conditions, but the underlying signal transduction mechanisms are incompletely understood. Increased DNA supercoiling and activation by potassium glutamate have each been proposed in alternative models as mediators of proU osmoresponsivity. We review here the available experimental data on proU regulation, and in particular the roles for DNA supercoiling, potassium glutamate, histone-like proteins of the bacterial nucleoid, and alternative sigma factors of RNA polymerase in such regulation. We also propose a new unifying model, in which the pronounced osmotic regulation of proU expression is achieved through the additive effects of at least three separate mechanisms, each comprised of a cis element [two promoters P1 and P2, and negative-regulatory-element (NRE) downstream of both promoters] and distinct trans-acting factors that interact with it: stationary-phase sigma factor RpoS with P1, nucleoid proteins HU and IHF with P2, and nucleoid protein H-NS with the NRE. In this model, potassium glutamate may activate proU expression through each of the three mechanisms whereas DNA supercoiling has a very limited role, if any, in the osmotic induction of proU transcription. We also suggest that proU may be a virulence gene in the pathogenic enterobacteria.
...
PMID:How is osmotic regulation of transcription of the Escherichia coli proU operon achieved? A review and a model. 908 63

Leukemia in the soft-shell clam, Mya arenaria, is characterized by tumor cells which are detected initially in the hemolymph. This disease is much more common in clams inhabiting polluted waters, suggesting an environmental component to its pathogenesis. In this study, leukemia cells were identified using a murine monoclonal antibody, 1E10, which recognizes a leukemia-specific protein expressed by tumor cells. Mutant p53 protein was detected using a murine monoclonal antibody (PAb 240) which reacts with mutant p53. Using immunofluorescence, the reactivity of clam cells to the 1E10 antibody was evaluated along with mutant p53 protein reactivity. Reverse transcriptase-polymerase chain reactions followed by sequence analyses were utilized to examine clams with hemocytes reacting with the p53 antibody for possible p53 gene mutations. Mutant p53 protein was expressed by tumor cells from five animals with advanced disease (in which greater than 90% of cells reacted with 1E10). A C-->G transversion was detected at the end of exon 6 from two of the five animals that reacted with both the mutant p53 antibody and 1E10. This substitution changes the amino acid of this codon from proline to alanine. Overall, our results suggest that environmentally induced alterations in p53 can contribute to the pathogenesis of leukemia in soft-shell clams inhabiting polluted water and/or sediment.
...
PMID:Detection of mutant p53 in clam leukemia cells. 916 98

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the sigma D form of RNA polymerase. The level of beta-galactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No beta-galactosidase could be detected in a sigD genetic background.
...
PMID:Functional and genetic characterization of mcpC, which encodes a third methyl-accepting chemotaxis protein in Bacillus subtilis. 935 24

We developed two methods for stable-isotope labeling of proteins by cell-free synthesis. Firstly, we applied cell-free synthesis to the dual amino acid-selective 13C-15N labeling method, originally developed for in vivo systems by Kainosho and co-workers. For this purpose, we took one of the advantages of a cell-free protein synthesis system; the amino acid-selective stable-isotope labeling is free of the isotope scrambling problem. The targets of selective observation were Thr35 and Ser39 in the effector region (residues 32-40) of the Ras protein complexed with the Ras-binding domain of c-Raf-1 (Raf RBD) (the total molecular mass is about 30 kDa). Using a 15-mL Escherichia coli cell-free system, which was optimized to produce about 0.4 mg of Ras protein per 1-mL reaction, with 2 mg each of DL-[13C']proline and L-[15N]threonine, we obtained about 6 mg of Ras protein. As the Pro-Thr sequence is unique in the Ras protein, the Thr35 cross peak of the Ras.Raf RBD complex was unambiguously identified by the 2D 1H-15N HNCO experiment. The Ser-39 cross peak was similarly identified with the [13C']Asp/[15N]Ser-selectively labeled Ras protein. There were no isotope scrambling problems in this study. Secondly, we have established a method for producing a milligram quantity of site-specifically stable-isotope labeled protein by a cell-free system involving amber suppression. The E. coli amber suppressor tRNATyrCUA (25 mg) was prepared by in vitro transcription with T7 RNA polymerase. We aminoacylated the tRNATyrCUA transcript with purified E. coli tyrosyl-tRNA synthetase, using 2 mg of L-[15N]tyrosine. In the gene encoding the Ras protein, the codon for Tyr32 was changed to an amber codon (TAG). This template DNA and the [15N]Tyr-tRNATyrCUA were reacted for 30 min in 30 mL of E. coli cell-free system. The subsequent purification yielded 2.2 mg of [15N]Tyr32-Ras protein. In the 1H-15N HSQC spectrum of the labeled Ras protein, only one cross peak was observed, which was unambiguously assigned to Tyr32.
...
PMID:Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. 969 Dec 77

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.
...
PMID:Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros. 973 15

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.
...
PMID:Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells. 986 47

3'-end formation is a complex and incompletely understood process involving both cis-acting and trans-acting factors. As part of an effort to examine the mechanisms of transcription termination by RNA polymerase II, a mutant hunt for strains defective in 3'-end formation was conducted. Following random mutagenesis, a temperature-sensitive strain exhibiting several phenotypes consistent with a role in transcription termination was isolated. First, readthrough of a terminator increases significantly in the mutant strain. Accordingly, RNA analysis indicates a decrease in the level of terminated transcripts, both in vivo and in vitro. Moreover, a plasmid stability assay in which high levels of readthrough lead to high levels of plasmid loss and transcription run-on analysis also demonstrate defective termination of transcription. Examination of polyadenylation and cleavage by the mutant strain indicates these processes are not affected. These results represent the first example of a transcription termination factor in Saccharomyces cerevisiae that affects transcription termination independent of 3'-end processing of mRNA. Complementation studies identified GRS1, an aminoacyl-tRNA synthetase, as the complementing gene. Sequence analysis of grs1-1 in the mutant strain revealed that nucleotides 1656 and 1657 were both C to T transitions, resulting in a single amino acid change of proline to phenylalanine. Further studies revealed GRS1 is essential, and the grs1-1 allele confers the temperature-sensitive growth defect associated with the mutant strain. Finally, we observed structures with some similarity to tRNA molecules within the 3'-end of various yeast genes. On the basis of our results, we suggest Grs1p is a transcription termination factor that may interact with the 3'-end of pre-mRNA to promote 3'-end formation.
...
PMID:A mutation in GRS1, a glycyl-tRNA synthetase, affects 3'-end formation in Saccharomyces cerevisiae. 1022 48

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.
...
PMID:Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation. 1053 63

ProP is an integral membrane transporter of proline, glycine betaine, and several other osmoprotecting compounds. Fis plus RpoS collaborate to promote a burst of proP transcription in late exponential growth phase. This brief period of ProP synthesis enables stationary phase cells to cope with a potential hyperosmotic shock. Fis activates the RpoS (sigma(38))-dependent proP P2 promoter by binding to a site within the promoter region centered at -41 and thus functions as a class II activator. We show here that activation by Fis at this promoter is completely dependent upon the alpha-CTD of RNA polymerase and that the activation domain on Fis is localized to a four amino acid ridge on the surface of Fis adjacent to the helix-turn-helix DNA binding domain in only one subunit of the homodimer. Fis mutants containing amino acid substitutions within this region are defective in cooperative binding interactions with the sigma(38)-form of RNA polymerase. Some of these substitutions also alter interactions with DNA sequences flanking the core binding site, but we show that changes in Fis-mediated curvature do not affect promoter activity. We conclude that the same amino acids are used by Fis to activate transcription from a class I (-71, rrnB P1) and class II (-41, proP P2) location, but this region is distinct from that required to regulate the Hin site-specific DNA inversion reaction.
...
PMID:Localization of amino acids required for Fis to function as a class II transcriptional activator at the RpoS-dependent proP P2 promoter. 1061 Jul 62


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---