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)

Eukaryotic RNA polymerases I and III share two distinct alpha-related subunits that show limited homology to the alpha subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of alpha-related subunits in eukaryotes, we have previously identified the alpha-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42+, which encodes the alpha-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42+ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42+ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct alpha-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.
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PMID:Isolation and characterization of the fission yeast gene rpa42+, which encodes a subunit shared by RNA polymerases I and III. 1062 57

Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5'-phosphate-dependent enzyme that specifically catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium. The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G. M., and Stadtman, T. C. (1998) J. Biol. Chem. 273, 30921-30926). We purified SCL from pig liver and determined its partial amino acid sequences. Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA. The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M(r) 47, 201). We also determined the sequence of the N-terminal region of putative human SCL. These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters. The recombinant mSCL overproduced in Escherichia coli was a homodimer with the subunit M(r) of 47,000. The enzyme was pyridoxal phosphate-dependent and highly specific to L-selenocysteine (the k(cat)/K(m) value for L-selenocysteine was about 4,200 times higher than that for L-cysteine). Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis. This is the first report of the primary structure of mammalian SCL.
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PMID:cDNA cloning, purification, and characterization of mouse liver selenocysteine lyase. Candidate for selenium delivery protein in selenoprotein synthesis. 1069 12

Follistatin (FS, an activin-binding protein) and activin A (homodimer of inhibin betaA chain) promote and inhibit cell proliferation in rat liver, respectively. The roles of activin AB (heterodimer of inhibin betaA and betaB) and activin B (homodimer of inhibin betaB) in rat liver have not been elucidated yet. In this study, we examined, by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, whether the levels of FS, inhibin betaA and betaB mRNAs change in the carbon tetrachloride induced rat liver regeneration model. The analysis was made in an hour-by-hour manner during the early stage of liver injury. There are 2 types of FS mRNA, FS-288 and FS-315, and the levels of both had begun to increase at 3 h, were maximal at 6 h, remained constant up to 12 h, and thereafter gradually decreased. The inhibin betaA mRNA had started to decline at 3 h, reached its lowest level at 6 h, partly returned at 12 h, and remained constant up to 48 h. The inhibin betaB mRNA level had begun to increase at 1 h, was maximal at 3 h, remained constant up to 24 h, and returned to the original level at 48 h. These results indicate that FS and activin A may act reciprocally in liver regeneration, and also suggest that activin AB and B may play roles in liver regeneration that differ from that of activin A.
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PMID:Expression of inhibin betaA, betaB and follistatin mRNAs in the carbon tetrachloride induced rat liver regeneration model. 1086 30

Full-length cDNAs for DNA ligase IV and the alpha and beta isoforms of DNA ligase III were cloned from Xenopus laevis to permit study of the genes encoding mitochondrial DNA ligase. DNA ligase III alpha and III beta share a common NH(2) terminus that encodes a mitochondrial localization signal capable of targeting green fluorescent protein to mitochondria while the NH(2) terminus of DNA ligase IV does not. Reverse transcriptase-polymerase chain reaction analyses with adult frog tissues demonstrate that while DNA ligase III alpha and DNA ligase IV are ubiquitously expressed, DNA ligase III beta expression is restricted to testis and ovary. Mitochondrial lysates from X. laevis oocytes contain both DNA ligase III alpha and III beta but no detectable DNA ligase IV. Gel filtration, sedimentation, native gel electrophoresis, and in vitro cross-linking experiments demonstrate that mtDNA ligase III alpha exists as a high molecular weight complex. We discuss the possibility that DNA ligase III alpha exists in mitochondria in association with novel mitochondrial protein partners or as a homodimer.
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PMID:Two forms of mitochondrial DNA ligase III are produced in Xenopus laevis oocytes. 1159 19

Flock house virus (FHV) is the best studied member of the Nodaviridae, a family of small, nonenveloped, isometric RNA viruses of insects and fish. Nodavirus genomes comprise two single-stranded positive-sense RNA segments (RNAs 1 and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp replicates both genomic RNAs and also generates a subgenomic RNA (RNA3) that is not encapsidated. Although genomic RNAs replicate through negative-sense intermediates, little is known about these RNAs or the details of the replication mechanism. Negative-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, have been detected in previous studies. In this study we detected dimers of RNAs 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected Drosophila cells, as well as from mammalian and yeast cells supporting FHV RNA replication. Characterization of these RNA species by RT-PCR and sequence determination showed that they contained head-to-tail junctions of FHV RNAs. RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication. To examine the template properties of these dimeric RNAs, we made corresponding cDNAs and transcribed them from a T7 promoter in mammalian cells constitutively expressing T7 RNA polymerase, together with RNA1 to provide the RdRp. Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated from complete or partial homodimer templates and from an RNA2-RNA3 heterodimer.
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PMID:Characterization and template properties of RNA dimers generated during flock house virus RNA replication. 1168 50

The bacteriophage T4 AsiA protein is a multifunctional protein that simultaneously acts as both a repressor and activator of gene expression during the phage life cycle. These dual roles with opposing transcriptional consequences are achieved by modification of the host RNA polymerase in which AsiA binds to conserved region 4 (SR4) of sigma(70), altering the pathway of promoter selection by the holoenzyme. The mechanism by which AsiA flips this genetic switch has now been revealed, in part, from the three-dimensional structure of AsiA and the elucidation of its interaction with SR4. The structure of AsiA is that of a novel homodimer in which each monomer is constructed as a seven-helix bundle arranged in four overlapping helix-loop-helix elements. Identification of the protein interfaces for both the AsiA homodimer and the AsiA-sigma(70) complex reveals that these interfaces are coincident. Thus, the AsiA interaction with sigma(70) necessitates that the AsiA homodimer dissociate to form an AsiA-SR4 heterodimer, exchanging one protein subunit for another to alter promoter choice by RNA polymerase.
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PMID:Flipping a genetic switch by subunit exchange. 1174 91

Fis is a versatile transactivator that functions at many different promoters. Fis activates transcription at the RpoS-dependent proP P2 promoter when bound to a site that overlaps the minus sign35 hexamer by a mechanism that requires the C-terminal domain of the alpha subunit of RNA polymerase (alphaCTD). The region on Fis responsible for activating transcription through the alphaCTD has been localized to a short beta-turn near the DNA-binding determinant on one subunit of the Fis homodimer. We report here that Fis-dependent activation of proP P2 transcription requires two discrete regions on the alphaCTD. One region, consisting of residues 264-265 and 296-297, mediates DNA binding. A second patch, comprising amino acid residues 271-273, forms a ridge on the surface of the alphaCTD that we propose interacts with Fis. The accompanying paper shows that these same regions on alphaCTD are utilized for transcriptional activation at the rrnB and rrnE P1 promoters by Fis bound to a site upstream of the core promoter (centered at minus sign71/minus sign72). In addition to stimulation of proP P2 transcription by Fis, CRP co-activates this promoter when bound to a remote site upstream from the promoter (centered at -121.5). RNA polymerase preparations lacking one alphaCTD of the alpha dimer were employed to demonstrate that the beta'-associated alpha(II)CTD was utilized preferentially by Fis at proP P2 in the presence and absence of CRP. These experiments define the overall architecture of the proP P2 initiation complex where Fis and CRP each function through a different alphaCTD.
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PMID:The C-terminal domains of the RNA polymerase alpha subunits: contact site with Fis and localization during co-activation with CRP at the Escherichia coli proP P2 promoter. 1186 15

The holoenzyme formed by RNA polymerase II (RNAPII) and the Mediator complex is the target of transcriptional regulators in vivo. A three-dimensional structure of the yeast holoenzyme has been generated from electron microscopic images of single holoenzyme particles. Extensive changes in Mediator conformation required for interaction with RNAPII have been modeled by correlating the polymerase-bound and free Mediator structures. Determination of the precise orientation of the RNAPII in the holoenzyme indicates that Mediator contacts are centered on the RNAPII Rpb3/Rpb11 heterodimer, the eukaryotic homolog of the alpha(2) homodimer involved in transcription regulation in prokaryotes. Implications for the possible mechanism of transcription regulation by Mediator are discussed.
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PMID:Structure of the yeast RNA polymerase II holoenzyme: Mediator conformation and polymerase interaction. 1219 85

Overexpression of recombinant proteins in Escherichia coli often leads to a severe growth retardation of the host cells. The phage T7 promoter phi10 in a pET vector was utilized to express human superoxide dismutase. Induction with IPTG lead to an increase in protein content and cell size and a termination of cell division, due to the deviation of the general metabolic fluxes from all cellular processes to plasmid maintenance and foreign protein synthesis. To generate promoter mutants which are better tolerated by the host cells we constructed a random mutation library by PCR with degenerated primers in a part of the promoter involved in the binding to the RNA polymerase and the initiation of transcription. This library was sorted by flow cytometry for cells with a lower total protein content as an indicator for continued cell replication and hence a less severe stress situation. The clones obtained had a similar SOD production compared to the original strain, but were able to reach higher densities in a batch culture, which resulted in a higher total yield.
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PMID:Optimization of recombinant protein expression level in Escherichia coli by flow cytometry and cell sorting. 1220 90

Changes in gene expression regulated by peroxisome proliferator-activated receptor gamma (PPARgamma) and in gene expression related to the inhibin/activin-follistatin system in the rat testis induced by a single oral administration of di-n-butyl phthalate (DBP) (8.6 mmol/kg) were examined and compared with those in the control rats using reverse-transcriptase polymerase chain reaction (RT-PCR). The increase in cytochrome P450 4A1 mRNA, which is regulated by PPARalpha, was significant, but not so profound as the increase of P450 4A1 mRNA in the liver. In contrast, a remarkable increase in the mRNA level of plasminogen activator inhibitor-1 (PAI-1) was found in the testis, suggesting the activation of PPARgamma. The substantial increase in PAI-1 may be related to the disruption of spermatogenesis. On the other hand, significant suppression of the mRNA level of inhibin beta(B) and elevation in the mRNA level of follistatin, an activin-binding protein, were observed after the DBP-administration. Activin B, a homodimer of inhibin beta(B), is known to stimulate spermatogonial proliferation. The present results suggest that the suppression of spermatogenesis resulting from the changes in the expression of genes involved in the inhibin/activin-follistatin system is one of the mechanisms of the testicular atrophy induced by DBP.
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PMID:Changes in peroxisome proliferator-activated receptor gamma-regulated gene expression and inhibin/activin-follistatin system gene expression in rat testis after an administration of di-n-butyl phthalate. 1256 98


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