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 removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.
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PMID:A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors. 753 40

Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory diseases. In the present study, we investigated the potential role of NO in an ocular model of inflammation, endotoxin-induced uveitis (EIU), in Lewis rats. Injection of LPS in one footpad induces severe uveitis after 16 h, which is accompanied by an increase of NO in the aqueous and vitreous humors, as evaluated by nitrite assay. Reverse transcriptase-PCR experiments reveal a large increase of inducible NO synthase (iNOS) mRNA in the iris/ciliary body, from 2 to 24 h after LPS treatment. In the retina, maximal increase of iNOS mRNA was detected 16 h after LPS treatment. Two i.p. injections of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), which inhibits nitrite release in the aqueous and vitreous humors, profoundly reduce clinical and histologic inflammation in EIU rats. These results implicate the NO pathway in the pathogenesis of EIU and demonstrate the possibility of modulating this inflammatory disease by injection of a NOS inhibitor.
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PMID:Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase. 753 24

Dimers of P22 Arc repressor bind to half-sites of the 21 bp arc operator and interact cooperatively to stabilize a DNA-bound tetramer. Mutation of Ser35 (a residue in the dimer-dimer interface) to Arg or Leu disrupts cooperative binding. The mutant proteins have near wild-type stabilities, give operator footprints like wild-type, and prevent binding of RNA polymerase to the Pant promoter in vitro. These mutants are, however, largely inactive in vivo. Thus, although cooperativity is not structurally required for repression, it appears that the additional DNA-binding energy from dimer-dimer cooperativity is required for normal biological function. Altering the spacing between the DNA half-sites by even one base-pair eliminates dimer-dimer cooperativity, indicating that Arc dimers need to be oriented correctly by half-site binding to allow the interactions that stabilize the tetrameric complex.
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PMID:P22 Arc repressor: role of cooperativity in repression and binding to operators with altered half-site spacing. 760 85

We have overproduced the leader peptidase from Escherichia coli in a high yield by using a T7 RNA polymerase/promoter system and purified the enzyme. This leader peptidase showed an apparent pH optimum of about 10 toward a synthetic peptide substrate, and was stable at temperatures below 40 degrees C. Kinetic studies indicated that one of the active site residues in the enzyme has a pKa value of approximately 7.5. The enzyme was rapidly inactivated by reaction with N-bromosuccinimide (NBS). When approximately two tryptophan residues were oxidized with NBS, the activity was almost completely lost and this inactivation was markedly prevented by a substrate. These NBS-reactive tryptophan residues were identified as Trp300 and Trp310 by a peptide mapping analysis. This indicates that Trp300 and/or Trp310 are critically important for the activity of the leader peptidase. On the other hand, the enzyme was scarcely inhibited by treatment with N-acetylimidazole, iodoacetic acid, 5,5'-dithiobis(2-nitrobenzoic acid), succinic anhydride, or 2,4,6-trinitrobenzenesulfonate. Diethylpyrocarbonate inhibited the enzyme; however, this inhibition did not seem to result from the modification of histidine residues. Thus, there seem to be no functionally important tyrosine, cysteine, or histidine residues or amino groups among the residues which readily react with these reagents. However, the enzyme was inactivated significantly by treatment with phenylglyoxal or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Therefore, some of the arginine residues and the carboxyl groups appear to be important for the enzyme activity.
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PMID:Leader peptidase from Escherichia coli: overexpression, characterization, and inactivation by modification of tryptophan residues 300 and 310 with N-bromosuccinimide. 762 19

Mutations in the human XPD gene result in a defect in nucleotide excision repair of ultraviolet damaged DNA and cause the cancer-prone syndrome xeroderma pigmentosum (XP). Besides XP, mutations in XPD can cause another seemingly unrelated syndrome, trichothiodystrophy (TTD), characterized by sulfur-deficient brittle hair, ichthyosis, and physical and mental retardation. To ascertain the underlying defect responsible for TTD, we have expressed the TTD mutant proteins in the yeast Saccharomyces cerevisiae and determined if these mutations can rescue the inviability of a rad3 null mutation. RAD3, the S. cerevisiae counterpart of XPD, is required for nucleotide excision repair and also has an essential role in RNA polymerase II transcription. Expression of the wild type XPD protein or the XPD Arg-48 protein carrying a mutation in the DNA helicase domain restores viability to the rad3 null mutation. Interestingly, the XPD variants containing TTD mutations fail to complement the lethality of the rad3 null mutation, strongly suggesting that TTD mutations impair the ability of XPD protein to function normally in RNA polymerase II transcription. From our studies, we conclude that XPD DNA helicase activity is not essential for transcription and infer that TTD mutations in XPD result in a defect in transcription.
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PMID:Lethality in yeast of trichothiodystrophy (TTD) mutations in the human xeroderma pigmentosum group D gene. Implications for transcriptional defect in TTD. 762 61

Our long-term goal is to define the catalytic domains of the L protein subunit of the Sendai virus RNA polymerase. An aberrant polyadenylation phenotype in the vesicular stomatitis virus tsG16 L protein mutant has recently been identified as a phenylalanine to serine change at amino acid 1488 (Hunt and Hutchinson, Virology 193, 786-793, 1993). To test if functional domains are conserved in the L proteins of negative-strand RNA viruses, we attempted to create a similar polyadenylation defect in the Sendai virus L protein. Nine different amino acid substitutions at the analogous site in the Sendai L protein (cysteine at amino acid 1571) were constructed by site-directed mutagenesis of the gene. Each mutant L protein was synthesized and bound to the Sendai P protein to form the P-L polymerase complex. While none of these L mutants exhibited a change in polyadenylation, the single amino acid changes yielded a variety of activities in vitro. Mutants containing valine, leucine, or phenylalanine at amino acid 1571, amino acids found naturally in the L proteins of other paramyxoviruses, yielded polymerases that had biological activity equal to or better than the wild-type (WT) polymerase. Serine or threonine substitutions in the L protein at this position also resulted in polymerases with nearly WT synthetic activity. In contrast, a glycine substitution significantly decreased overall polymerase activity, whereas a tyrosine substitution gave decreased transcription, but virtually no DI genome replication in vitro. The tyrosine-substituted polymerase may be unable to carry out the packaging step of replication, since DI leader RNA synthesis was normal in this mutant. Mutant L proteins with basic arginine or histidine substitutions were inactive in all viral RNA synthesis in vitro, although the polymerase complexes could bind the nucleocapsid template.
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PMID:Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro. 764 61

Arg-104 of the kinase domain of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was mutated to alanine, the mutant enzyme expressed in E. coli with a T7 RNA polymerase-based expression system, and purified to homogeneity by Blue-Sepharose and Q-Sepharose chromatography. The mutant enzyme exhibited a 200-fold increase in Km for fructose-6-phosphate, no change in Km for ATP, and a 2-3-fold increase in catalytic rate. The results indicate that Arg-104, along with Arg-195, are the principal binding site residues for the 6-phosphate group of fructose-6-phosphate. In contrast to the corresponding residue in the related E. coli 6-phosphofructo-1-kinase, Arg-104 did not stabilize the transition state at pH 7-9. The Arg-104 mutation also decreased Fru-2,6-P2ase activity without affecting substrate inhibition, which suggests that this mutation affects the bisphosphatase active site conformation and/or substrate access to it.
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PMID:Evolutionary reengineering of the phosphofructokinase active site: ARG-104 does not stabilize the transition state in 6-phosphofructo-2-kinase. 764 23

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.
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PMID:Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. 767 94

A new platelet alloantigen, termed CA, has recently been implicated in a case of neonatal alloimmune thrombocytopenia (NATP) in a Filipino family in Canada. Maternal anti-CA serum reacted with glycoprotein (GP) IIIa and maintained its reactivity after removal of high mannose carbohydrate residues from GPIIIa. The monoclonal antibody (MoAb) AP3 partially blocked binding of anti-CA to GPIIIa, suggesting that the CA polymorphism is proximal to the AP3 epitope. Platelet RNA polymerase chain reaction (PCR) was used to amplify the region of GPIIIa cDNA that encodes this region of the protein. DNA sequence analysis showed a G<==>A nucleotide substitution at base 1564 that results in an arginine (Arg) (CGG)<==>glutamine (Gln) (CAG) polymorphism in amino acid (AA) 489. Further analysis of PCR-amplified genomic DNA from 27 normal individuals showed that AA 489 is encoded by a mutational "hot spot" of the GPIIIa gene, as three different codons for the wild-type Arg489 of GPIIIa were also found. The codon usage for Arg489 was found to be: CGG (63%), CGA (37%), and CGC (< 1%). These frequency data were valuable in determining the relationship of the CA alloantigen to the serologically defined TU GPIIIa polymorphism that is present in low frequency in the Finish population. Analyses of PCR-amplified genomic DNA showed the CA and TU alloantigens to be identical at the molecular level. Definition of these new molecular variants of the beta 3 integrin chain should prove valuable in the diagnosis of NATP in these two geographically disparate populations, and it may also provide useful genetic markers for examining other pathologic variations of the GPIIb-IIIa complex.
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PMID:Amino acid 489 is encoded by a mutational "hot spot" on the beta 3 integrin chain: the CA/TU human platelet alloantigen system. 769 83

We have identified a temperature-sensitive mutant of Saccharomyces cerevisiae (npl3) that accumulates polyadenylated RNA in the nucleus at 37 degrees C, as judged by in situ hybridization. The strong nuclear signal is not simply due to increased cytoplasmic turnover of mRNA, as reincubation at 37 degrees C with an RNA polymerase inhibitor shows no diminution in the in situ signal. Over several hours at 37 degrees C, the average poly(A) tail length increases and a characteristic ultrastructural alteration of the nucleoplasm occurs. Cloning and sequencing indicate that the corresponding gene is NPL3/NOP3, which codes for a nucleolar/nuclear protein implicated in protein import into the nucleus (Bossie et al. (1992). Mol. Biol. Cell 3, 875-893) and in rRNA maturation (Russell and Tollervey (1992). J. Cell Biol. 119, 737-747). NPL3 includes bipartite RNA recognition motifs (RRM) and a Gly-Arg repeat domain, as in several nucleolar proteins. A point mutation adjacent to one of the RRM has been identified in the ts copy of the gene. Although this protein is not concentrated in nuclear pores, NPL3 is implicated in both import and export from the nucleus. Judging from the site of the npl3 mutation and since the block in RNA export can be detected prior to an obvious nuclear import defect in npl3, the defect in RNA export may be primary. Since other mutants that interrupt RNA export do not block protein import, the NPL3 protein itself appears to be implicated in protein import.
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PMID:A yeast protein that bidirectionally affects nucleocytoplasmic transport. 773 4


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