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)

Biological polyadenylation, first recognized as an enzymatic activity, remained an orphan enzyme until poly A sequences were found on the 3' ends of eukarvotic mRNAs. Their presence in bacteria viruses and later in archeae (ref. 338) established their universality. The lack of compelling evidence for a specific function limited attention to their cellular formation. Eventually the newer techniques of molecular biology and development of accurate nuclear processing extracts showed 3' end formation to be a two-step process. Pre-mRNA was first cleaved endonucleolytically at a specific site that was followed by sequential addition of AMPs from ATP to the 3' hydroxyl group at the end of mRNA. The site of cleavage was specified by a conserved hexanucleotide, AAUAAA, from 10 to 30 nt upstream of this 3' end. Extensive purification of these two activities showed that more than 10 polypeptides were needed for mRNA 3' end formation. Most of these were in complexes involved in the cleavage step. Two of the best characterized are CstF and CPSF, while two other remain partially purified but essential. Oddly, the specific proteins involved in phosphodiester bond hydrolysis have yet to be identified. The polyadenylation step occurs within the complex of poly A polymerase and poly A-binding protein, PABII, that controls poly A length. That the cleavage complex, CPSF, is also required for this step attests to a tight coupling of the two steps of 3' and formation. The reaction reconstituted from these RNA-free purified factors correctly processes pre-mRNAs. Meaningful analysis of the role of poly A in mRNA metabolism or function was possible once quantities of these proteins most often over-expressed from cDNA clones became available. The large number needed for two simple reactions of an endonuclease, a polymerase and a sequence recognition factor, pointed to 3' end formation as a regulated process. Polyadenylation itself had appeared to require regulation in cases where two poly A sites were alternatively processed to produce mRNA coding for two different proteins. The 64-KDa subunit of CstF is now known to be a regulator of poly A site choice between two sites in the immunoglobulin heavy chain of B cells. In resting cells the site used favors the mRNA for a membrane-bound protein. Upon differentiation to plasma cells, an upstream site is used the produce a secreted form of the heavy chain. Poly A site choice in the calcitonin pre-mRNA involves splicing factors at a pseudo splice site in an intron downstream of the active poly site that interacts with cleavage factors for most tissues. The molecular basis for choice of the alternate site in neuronal tissue is unknown. Proteins needed for mRNA 3' end formation also participate in other RNA-processing reactions: cleavage factors bind to the C-terminal domain of RNA polymerase during transcription; splicing of 3' terminal exons is stimulated port of by cleavage factors that bind to splicing factors at 3' splice sites. nuclear ex mRNAs is linked to cleavage factors and requires the poly A II-binding protein. Most striking is the long-sought evidence for a role for poly A in translation in yeast where it provides the surface on which the poly A-binding protein assembles the factors needed for the initiation of translation. This adaptability of eukaryotic cells to use a sequence of low information content extends to bacteria where poly A serves as a site for assembly of an mRNA degradation complex in E. coli. Vaccinia virus creates mRNA poly A tails by a streamlined mechanism independent of cleavage that requires only two proteins that recognize unique poly A signals. Thus, in spite of 40 years of study of poly A sequences, this growing multiplicity of uses and even mechanisms of formation seem destined to continue.
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PMID:A history of poly A sequences: from formation to factors to function. 1210 57

We have studied the expression of 1L-myoinositol-1-phosphate synthase (MIPS; EC 5.5.1.4) in developing organs of Phaseolus vulgaris to define genetic controls that spatially regulate inositol phosphate biosynthesis. MIPS, the pivotal biosynthetic enzyme in inositol metabolism, is the only enzyme known to catalyze the conversion of glucose 6-phosphate to inositol phosphate. It is found in unicellular and multicellular eukaryotes and has been isolated as a soluble enzyme from both. Thus, it is widely accepted that inositol phosphate biosynthesis is largely restricted to the cytosol. Here, we report findings that suggest the enzyme is also expressed in membrane-bound organelles. Microscopic and biochemical analyses detected MIPS expression in plasma membranes, plastids, mitochondria, endoplasmic reticula, nuclei, and cell walls of bean. To address mechanisms by which the enzyme could be targeted to or through membranes, MIPS genes were analyzed for sorting signals within primary structures and upstream open reading frames that we discovered through our sequence analyses. Comprehensive computer analyses revealed putative transit peptides that are predicted to target the enzyme to different cellular compartments. Reverse transcriptase PCR experiments suggest that these putative targeting peptides are expressed in bean roots and leaves.
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PMID:Expression of 1L-myoinositol-1-phosphate synthase in organelles. 1291 78

The authors studied mineralocorticoid receptor (MCR)-mediated effects of steroids on CD34(+) progenitor cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed the presence of mRNA for both the MCR and the alpha subunit of the epithelial sodium channel, a member of the amiloride-sensitive sodium channel (ASSC) superfamily, in human CD41(+) megacaryoblastic cells derived from cultured bone marrow CD34(+) isolates, as well as in the human erythromegakaryoblastic leukemia (HEL) cell line. Immunofluorescence also revealed the presence of both the MCR and ASSC in circulating CD34(+) and medullar CD41(+) megacaryoblastic cells, the former as a nucleocytoplasmic protein and the latter as a membrane-bound protein, as expected from earlier studies using MCR-specific targets. In a selective medium, the formation of erythrocyte burst-forming units, and of the granulocyte-macrophage colony-forming units, by circulating CD34(+) cells was influenced by the agonists deoxycorticosterone and aldosterone, as well as by the antagonists RU 26752 and ZK 91587, targeted for the MCR. The multiplication of the leukemic HEL progeny, derived from CD41(+) cells, was similarly altered by these steroids targeted for the MCR. In contrast, in the optimal growth medium, the multiplication, and colony formation by bone marrow CD34(+) progenitor cells were not altered by either aldosterone or ZK 91587. These and other studies reveal that the receptor-mediated action of mineralocorticoids may influence the functional maturation of the hematopoietic progenitor lineage, contrary to the classical notion where the mineralotropic effect would be a unique feature of the epithelial cell.
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PMID:Mineralocorticoid hormones exert dramatic effects on pluripotent human stem cell progeny. 1293 24

The RNA polymerase sigma factor sigma(F) is a developmental regulatory protein that is activated in a cell-specific manner following the formation of the polar septum during the process of spore formation in the bacterium Bacillus subtilis. Activation of sigma(F) depends on the membrane-bound phosphatase SpoIIE, which localizes to the septum, and on the formation of the polar septum itself. SpoIIE is responsible for dephosphorylating and thereby activating the phosphoprotein SpoIIAA, which, in turn, triggers the release of sigma(F) from the anti-sigma(F) factor SpoIIAB. Paradoxically, however, the presence of unphosphorylated SpoIIAA is insufficient to cause sigma(F) activation as SpoIIAA reaches substantial levels in mutants blocked in polar septation. We now describe mutants of SpoIIE, SpoIIAA, and SpoIIAB that break the dependence of sigma(F) activation on polar division. Analysis of these mutants indicates that unphosphorylated SpoIIAA must reach a threshold concentration in order to trigger the release of sigma(F) from SpoIIAB. Evidence is presented that this threshold is created by the action of SpoIIAB, which can form an alternative, long lived complex with SpoIIAA. We propose that formation of the SpoIIAA-SpoIIAB complex serves as a sink that traps SpoIIAA in an inactive state and that only when unphosphorylated SpoIIAA is in excess to the sink does activation of sigma(F) take place.
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PMID:A threshold mechanism governing activation of the developmental regulatory protein sigma F in Bacillus subtilis. 1474 53

The 60 nucleotides encoding the signal peptide of the Neisseria meningitidis membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of GNA33 from either a T7- or a P(lac)-driven system in Escherichia coli. Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem-loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the GNA33 transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.
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PMID:The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression. 1513 4

TATA binding protein (TBP) is a central transcription factor used by all three cellular RNA polymerases. Changes in the levels of TBP have been shown to have selective effects on gene activity. Overexpression of TBP has been recently shown to contribute to cellular transformation, and elevated levels of TBP occur in a clinically significant proportion of human colon tumors relative to matched normal tissue. To understand the mechanisms by which TBP is regulated, we have analyzed whether activation of the epidermal growth factor receptor (EGFR), a membrane-bound tyrosine receptor kinase that is activated in a large number of human cancers, can serve to regulate cellular TBP. We show that treatment of mouse epidermal cells with EGF produces an increase in TBP levels, which can be blocked with an EGFR-specific inhibitor. In contrast, TBP levels remain unchanged after EGF treatment of EGFR null cells. EGF-mediated increases in TBP are regulated at the transcriptional level, as transient expression of the human TBP promoter is induced with EGF. This regulatory event is dependent upon the downstream activation of Ras and requires the activation of p38, JNK, and ERK mitogen-activated protein kinases. The consequence of elevated TBP on gene expression was further determined. Transcription by RNA polymerase (Pol) I and III was induced by EGF. Directly overexpressing TBP also stimulated transcription from these promoters. Thus, we have identified a new and important target of EGFR signaling, TBP, that contributes to EGF-mediated stimulation of RNA Pol I- and III-dependent gene activity. Since the cellular levels of the products of these genes, tRNAs and rRNAs, determine the translational capacity of cells, this event may be an important contributor to the transforming function of EGF.
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PMID:Epidermal growth factor enhances cellular TATA binding protein levels and induces RNA polymerase I- and III-dependent gene activity. 1516 79

The Des pathway of Bacillus subtilis regulates the synthesis of the cold-shock induced membrane-bound enzyme Delta5-fatty acid desaturase (Delta5-Des). A central component of the Des pathway is the response regulator, DesR, which is activated by a membrane-associated kinase, DesK, in response to a decrease in membrane lipid fluidity. Despite genetic and biochemical studies, specific details of the interaction between DesR and the DNA remain unknown. In this study we show that only the phosphorylated form of protein DesR is able to bind to a regulatory region immediately upstream of the promoter of the Delta5-Des gene (Pdes). Phosphorylation of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P DNA binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator. Subsequently, this phosphorylation signal propagation leads to the activation of the des gene through recruitment of RNA polymerase to Pdes. This is the first dissected example of a transcription factor functioning as a phosphorylation-activated switch for a cold-shock gene, allowing the cell to optimize the fluidity of membrane phospholipids.
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PMID:Bacillus subtilis DesR functions as a phosphorylation-activated switch to control membrane lipid fluidity. 1524 25

Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3' flanking region (rs165599)--both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val--had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3' SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a predominant factor that determines higher COMT activity in the prefrontal cortex, which presumably leads to lower synaptic dopamine levels and relatively deleterious prefrontal function.
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PMID:Functional analysis of genetic variation in catechol-O-methyltransferase (COMT): effects on mRNA, protein, and enzyme activity in postmortem human brain. 1545 4

Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.
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PMID:ADP-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture. 1618 75

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metalloprotease responsible for production of vasoactive endothelin (ET)-1 from inactive big ET-1. ECE-1 exists as four separate isoforms, ECE-1a, b, c, and d, which differ only in their amino-terminal regions. We investigated the expression and localization of the ECE-1 isoforms in primary human umbilical vein endothelial cells (HUVECs) and EAhy926 cells. Reverse transcriptase polymerase chain reaction showed expression of all four isoforms in both cell lines, with ECE-1d seeming, at least qualitatively, to be the predominant isoenzyme. Isoform-specific polyclonal antibodies were used to investigate isoform protein expression. ECE-1a, b, and c protein was detected in EAhy926 cells by immunoblotting; only ECE-1a and ECE-1c were detected in HUVECs. Using immunofluorescence microscopy analysis, both HUVEC and EAhy926 cells showed nuclear immunoreactivity with a monoclonal antibody recognizing all ECE-1 isoforms. The ECE-1a antibody also showed nuclear immunoreactivity in both cell lines; this seemed to colocalize with nucleolin. The ECE-1b antibody showed nuclear immunoreactivity in EAhy926 cells, but no overlap with nucleolin was seen. Intracellular immunoreactivity was seen in both cell lines using the ECE-1c antibody; this showed some colocalization with concanavalin A (an endoplasmic reticulum marker). von Willebrand Factor was used as a marker for Weibel-Palade bodies in HUVECs, but no colocalization with ECE-1 was seen during this study. The data presented here sheds new light on the localization of ECE-1a, b, and c in cultured human endothelial cells, which may further understanding of the ET system and aid design of therapeutic ECE inhibitors.
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PMID:Expression and localization of endothelin-converting enzyme-1 isoforms in human endothelial cells. 1674 Sep 87


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