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 co-infection or infection-transfection variants of the T7 RNA polymerase/vaccinia vector system were used to express 5-HT1ARs in COS-7, BSC-40 and GH3 cells, with co-infection giving ca. 3-fold higher level than infection-transfection. Binding affinities were similar to those of the endogenous 5-HT1AR, with highest affinities for 5-HT and 8-OH-DPAT. Functional properties were demonstrated by assays of agonist-stimulated GTPase activity and its inhibition by pertussin toxin. Immunoblot assays showed expression of the unglycosylated and glycosylated receptor protein in the membrane and, surprisingly, in the cytosolic fractions.
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PMID:Expression in animal cells of the 5-HT1A receptor by a vaccinia virus vector system. 153 96

We recently identified a novel rat cDNA: rab1B, closely related to the rab1A cDNA and to the yeast YPT1 gene. The rab1B cDNA encodes a 202 amino acid protein (22.1 kDa) that was produced in Escherichia coli under the control of the phi 10 promoter for the T7 RNA polymerase. The rab1B protein was purified in large amounts to near homogeneity in a simplified procedure. We studied the biochemical properties of rab1B and rab1A proteins. They both bind specifically GTP and GDP and possess intrinsic GTPase activities. The rab1B Lys21----Met mutant protein does not bind GTP, whereas the Ala65----Thr mutant has a reduced GTPase activity and is competent for autophosphorylation in the presence of GTP.
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PMID:Biochemical properties of the YPT-related rab1B protein. Comparison with rab1A. 250 43

Poliovirus infection leads to drastic alterations in membrane permeability late during infection. Transient expression of each nonstructural protein of poliovirus by means of recombinant vaccinia virus encoding the T7 RNA polymerase indicates that proteins 2B and 2BC strongly enhance membrane permeability to hygromycin B in HeLa cells. Almost no effect on expression of proteins 2C, 3A, 3AB, and 3C was found. Deletions and point mutations in 2B and 2BC have identified sequences in 2B involved in membrane permeabilization. Regions located at both ends of 2B are necessary to bring about these permeability alterations. A deletion of 11 amino acids of 2BC at the junction between 2B and 2C, as well as long deletions in 2C encompassing the GTPase motifs of this protein, do not impair the capacity of 2BC to modify the permeability of the membrane. The release of compounds such as choline or uridine from preloaded cells is also augmented by 2B and 2BC expression.
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PMID:Membrane permeabilization by poliovirus proteins 2B and 2BC. 879 6

The elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was expressed in Escherichia coli and purified. The SsEF-1 alpha gene was amplified by PCR and cloned in the Ndel site of the pT7-7 expression vector, under the control of the promoter of T7 RNA polymerase. Upon induction with isopropyl beta-D-thiogalactopyranoside, the recombinant SsEF-1 alpha (recSsEF-1 alpha) was purified from the E. coli S-100 extract by a two-step procedure. From 1 litre of cell culture, about 2 mg of purified recSsEF-1 alpha was obtained. The N-terminal sequence of the first 30 amino acid residues of recSsEF-1 alpha was identical with that translated from the nucleotide sequence of the corresponding gene, except for the initial residue, which in recSsEF-1 alpha was Ser instead of Met. The M(r) of recSsEF-1 alpha (determined by electrospray MS) was almost coincident with that of the naturally occurring SsEF-1 alpha (SsEF-1 alpha). The thermal-inactivation and thermophilicity profiles of SsEF-1 alpha and recSsEF-1 alpha were identical. Concerning the functional properties, recSsEF-1 alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to elicit an NaCl-dependent GTPase activity [Masullo, De Vendittis and Bocchini (1994) J. Biol. Chem. 269, 20376-20379] with the same efficiency as that displayed by SsEF-1 alpha.
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PMID:Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus. 886 95

The expression of genes transcribed by the RNA polymerase with the alternative sigma factor sigma 54 (E sigma 54) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter. These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis. The best characterized proteins of this family of regulators are NtrC and NifA, which activate genes required for ammonia assimilation and nitrogen fixation, respectively. In a recent IRBM course (@ontiers of protein structure prediction," IRBM, Pomezia, Italy, 1995; see web site http://www.mrc-cpe.cam.uk/irbm-course95/), one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold. This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology. In this work, we present new data that support the previous conclusion. The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alpha/beta topology. The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence. Finally, and most importantly, three of five reported residue alterations that impair the Central domain. ATPase activity of the E sigma 54 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotide-binding in the mononucleotide-binding proteins. Furthermore, the known residue substitution that alter the function of the E sigma 54 activators, leaving intact the Central domain ATPase activity, are mapped on region proposed to play an equivalent role as the effector region of the GTPase superfamily.
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PMID:A proposed architecture for the central domain of the bacterial enhancer-binding proteins based on secondary structure prediction and fold recognition. 907 Apr 37

The plant-specific Rop family GTPases are versatile molecular switches in many processes during plant growth, development, and responses to the environment. To understand how Rop achieves its functional versatility in signaling, we performed a genome-wide identification of putative Rop targets using a combination of the yeast two-hybrid method, bioinformatic tools, and a robust functional assay in pollen. In this study, we have identified 11 Arabidopsis genes encoding novel proteins, termed RICs (for Rop-interactive CRIB motif-containing proteins), that contain a CRIB (for Cdc42/Rac-interactive binding) motif required for their specific interaction with GTP-bound Rop1. RICs are divergent and classified into five groups that share little sequence homology outside of the conserved Rop-interactive domain. Overexpression in tobacco pollen tubes of the nine Ric genes that are expressed in Arabidopsis pollen causes distinct phenotypes, implying distinct functions for various RICs. RIC3 (group III) and RIC4 (group V) both cause depolarized growth like Rop1 and display Rop1-enhanced localization to the tip of pollen tubes, suggesting that these RICs may be two distinct targets of Rop1. In contrast, RIC10 (group I) promotes pollen tube elongation but does not affect pollen tube growth polarity and shows Rop1-independent localization to the cytoplasm, suggesting that RIC10 may participate in a Rop1-independent pathway probably controlled by a different Rop. Expression of all other RICs causes various degrees of growth inhibition in pollen tubes. Furthermore, these inhibitory RICs also exhibit distinct patterns of localization in pollen tubes. Our results suggest that various RICs have evolved to interact with Rops differentially and to perform distinct functions in pollen tubes. Reverse transcriptase-mediated polymerase chain reaction analysis showed that six of the nine RICs are expressed in various parts of Arabidopsis plants. On the basis of these observations, we propose that RICs function as Rop GTPase targets that control various Rop-dependent signaling pathways in plants.
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PMID:A genome-wide analysis of Arabidopsis Rop-interactive CRIB motif-containing proteins that act as Rop GTPase targets. 1175 91

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3' to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the DeltaafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation of afsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues.
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PMID:afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2). 1195 95

Transcriptional activator proteins that act upon the sigma54-containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone-like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with sigma54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the creation of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with sigma54-holoenzyme occur as a consequence of sensing the state of the gamma-phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone-like binding activities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase-activating proteins.
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PMID:Mechanochemical ATPases and transcriptional activation. 1218 Sep 11

In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.
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PMID:Towards a phosphoproteome map of Corynebacterium glutamicum. 1292 88

Yeast Gtr1p and its human homolog RRAG A belong to the Ras-like small G-protein superfamily and genetically interact with RCC1, a guanine nucleotide exchange factor for Ran GTPase. Little is known regarding the function of Gtr1p. We performed yeast two-hybrid screening using Gtr1p as the bait to find interacting proteins. Rpc19p, a shared subunit of RNA polymerases I and III, associated with Gtr1p. The association of Gtr1p with Rpc19p occurred in a GTP-form-specific manner. RRAG A associated with RPA16 (human Rpc19p homolog) in a GTP-form-specific manner, suggesting that the association is conserved during evolution. Ribosomal RNA and tRNA synthesis were reduced in the gtr1Delta strain expressing the GDP form of Gtr1p, but not the GTP form of Gtr1p. Gel-filtration studies revealed an accumulation of the smaller Rpc19p-containing complex, but not of A135, in the gtr1Delta strain. Here, we propose that Gtr1p is involved in RNA polymerase I and III assembly by its association with Rpc19p and could be a mediator that links growth regulatory signals with ribosome biogenesis.
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PMID:Association of the GTP-binding protein Gtr1p with Rpc19p, a shared subunit of RNA polymerase I and III in yeast Saccharomyces cerevisiae. 1593 28


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