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 C-terminal domain (CTD) of RNA polymerase II (RNAP) has an essential function in the regulation of transcription. The CTD of the human malaria parasite, Plasmodium falciparum, differs dramatically from that of higher eukaryotes. To determine whether this is a general feature of malarial parasites, we have analysed the CTD of the distantly related rodent malaria parasite P.berghei. The CTDs of the two parasites enzymes are very similar in amino acid composition and contain the basic structure of most eukaryotic CTDs, which is a tandem repeat of a heptapeptide (SPTSPSY). The CTD of P.berghei differs, however, in three aspects from the CTD of P.falciparum and other eukaryotes. First, both domains show a divergence from the consensus sequence at position 6 of the heptapeptide repeat. The Ser6 is always substituted, with a bias for lysine. The latter substitution might increase the binding efficiency to the DNA template. Second, the rodent and human malarial CTDs contain a 3' extension of, respectively, 66 or 67 amino acid residues. This tail-piece is unique among eukaryotes. Third, the enlargement of the CTD of the human parasite by six heptapeptide repeats is most likely generated by a recent amplification of a specific repeat unit.
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PMID:The C-terminal domain of RNA polymerase II of the malaria parasite Plasmodium berghei. 184 Apr 89

A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [alpha-32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue. Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue. Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity. For the Lys----Gly mutant enzyme, anomalous template binding was observed.
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PMID:Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis. 184 71

The S1 segments, encoding the group-specific antigen, VP7, from the five United States prototype BTV serotypes were cloned as full-length entities. The nucleotide and deduced amino acid sequences of segment S1 of BTV-2 were determined and compared with BTV-10, -11, -13, and -17, completing the sequencing of this cognate gene segment from all five US BTV serotypes. Each segment is 1156 bp long and contains an open reading frame encoding the 349-amino acid VP7 protein. Most (greater than 94%) of the amino acids of VP7 among the serotypes are conserved, including the location (position 255) of a single lysine residue. Secondary structure analyses of VP7 predict a putative eight-stranded beta-barrel between amino acid positions 150 and 250, a structure similar to that observed in ssRNA viruses. The S1 genes are flanked by conserved 5' and 3' noncoding regions. Stem-loop structures are predicted at the 3' end of each gene (nucleotide positions 1058-1097). The S1 segments of BTV-2, -10, -11, and -17 have greater than 93% of the nucleotides conserved, while less than 80% of their bases are identical with BTV-13. Analyses of nucleotide mismatches in each codon position of the VP7 open reading frame, transition frequencies, and evolutionary distances show that of the five, BTV-13 is the most distantly related and that BTV-10 and -17 are the most closely related serotypes. Evolutionary distance calculations of segment L2 from BTV-10, -11, and -17 concur with these observations. Comparison of this relationship with hybridization data of segment M3, which codes for VP5, suggests that BTV-17 has evolved by a combination of genetic drift and genomic reassortment. The data also indicate that the five US BTV serotypes are derived from two distinct gene pools. Evolution distances were used to estimate an evolution rate of 2.2 x 10(-3) nucleotide substitution/site/year for BTV segment S1. This rate is similar to the genes of retroviruses and implies an absence of RNA polymerase proofreading activity for dsRNA viruses.
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PMID:Bluetongue virus evolution: sequence analyses of the genomic S1 segments and major core protein VP7. 184 84

The importance of certain amino acid residues in mammalian ornithine decarboxylase activity and degradation was studied by site-specific mutagenesis. Changes were made to the mouse ornithine decarboxylase cDNA in a plasmid containing a T7 RNA polymerase promoter. The plasmid was then used for the synthesis of RNA, which was translated in a reticulocyte lysate system. The activity of the ornithine decarboxylase formed and the stability of the protein to degradation in a reticulocyte lysate system were determined. Changes of lysine-169 or of histidine-197 to alanine completely abolished enzyme activity, indicating that these residues are essential for enzyme activity. The removal of the C-terminal 36 residues, the mutation of lysine-349 to alanine, of lysine-298 to alanine or the double change of serine-303 and glutamic acid-308 to alanine residues still resulted in an active enzyme. The last-mentioned finding indicates that the phosphorylation of serine-303 does not play an essential role in the catalytic activity of ornithine decarboxylase. The control ornithine decarboxylase protein was degraded rapidly in a reticulocyte lysate provided that ATP was added. The truncated protein missing the 36 residues from the C-terminus was much more stable in this system, and the protein containing the double change of serine-303 and glutamic acid-308 to alanine residues was slightly more stable than control ornithine decarboxylase protein. These results indicate that the altered residues may play a role in interaction with factors responsible for the rapid turnover of ornithine decarboxylase.
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PMID:Identification of residues in ornithine decarboxylase essential for enzymic activity and for rapid protein turnover. 187 2

ORF 10 of the K2 plasmid from Kluyveromyces lactis encodes a small basic protein (22.3% lysine). The function of its product has been investigated. Western blot analysis, using an antibody against MS2 RNA polymerase/ORF 10 fusion protein, reveals a protein band with an apparent molecular weight of 14 kDa. The protein can bind a DNA-Sepharose column, and is eluted by 350 mM-salt. Immunoprecipitation experiments show that the ORF 10 protein coprecipitates with the linear genomic DNAs of the two killer plasmids (K1 and K2). From Western/Southern blot data, it is possible to conclude that the interaction between protein and DNA occurs directly, rather than via other protein(s). ORF 10 is easily detected by Western blot and its transcript is one of the most abundant of the K2 plasmid, suggesting that this protein may have a structural rather than a regulatory function. This possibility is also suggested by the observed sequence homology between the ORF 10 protein and the family of histone-like proteins.
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PMID:Killer system of Kluyveromyces lactis: the open reading frame 10 of the pGK12 plasmid encodes a putative DNA binding protein. 188 49

To identify functionally important regions of the human interferon (IFN)-alpha molecule, mutagenesis in vitro of human IFN-a genes was used to create analogs with deletions or specific amino acid replacements. These analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate protein synthesis system. Deletion of 7 highly conserved hydrophilic amino acids from the C-terminus of human IFN-alpha 4 reduced, but did not abolish, antiviral activity on human cells. However, analogs with deletions of 15 or 25 amino acids from the C-terminus, or 28 amino acids from the N-terminus, had no measurable antiviral activity. The antiviral activity of human IFN-alpha 4 was increased by substitution of cysteine for serine at position 86, and lysine for arginine at position 121. However, other amino acid substitutions at positions 121, 122 or 123 reduced antiviral activity. The size of the side chain of the amino acid residue at position 130 was shown to be important. Replacement of the absolutely conserved leucine residue at position 131 with glutamine had little effect on antiviral activity. However, the introduction of a proline residue at this position abolished antiviral activity, probably due to the formation of a beta turn in the polypeptide chain. The antiviral activity of human IFN-alpha 4 on murine cells was increased by substitutions at positions 86, 121 and 133. This study illustrates the utility of the in vitro mutagenesis and rabbit reticulocyte lysate systems for the investigation of structure-function relationships, and extends our knowledge of the biologically active regions and species specificity of the human IFN-alpha molecule.
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PMID:Structure-function studies of human interferons-alpha: enhanced activity on human and murine cells. 190 22

Upon infection of animal cells by Sindbis virus, four nonstructural (ns) proteins, termed nsP1-4 in order from 5' to 3' in the genome, are produced by posttranslational cleavage of a polyprotein. nsP4 is believed to function as the viral RNA polymerase and is short-lived in infected cells. We show here that nsP4 produced in reticulocyte lysates is degraded by the N-end rule pathway, one ubiquitin-dependent proteolytic pathway. When the N-terminal residue of nsP4 is changed by mutagenesis, the metabolic stabilities of the mutant nsP4s follow the N-end rule, in that the half-life of nsP4 bearing different N-terminal residues decreases in the order Met greater than Ala greater than Tyr greater than or equal to Phe greater than Agr. Addition of dipeptides Tyr-Ala, Trp-Ala, or Phe-Ala to the translation mixture inhibits degradation of Tyr-nsP4 and Phe-nsP4, but not of Arg-nsP4. Conversely, dipeptides His-Ala, Arg-Ala, and Lys-Ala inhibit the degradation of Arg-nsP4 but not of Tyr-nsP4 or Phe-nsP4. We found that there is no lysine in the first 43 residues of nsP4 that is required for its degradation, indicating that a more distal lysine functions as the ubiquitin acceptor. Strict control of nsP4 concentration appears to be an important aspect of the virus life cycle, since the concentration of nsP4 in infected cells is regulated at three levels: translation of nsP4 requires read-through of an opal termination codon such that it is underproduced; differential processing by the virus-encoded proteinase results in temporal regulation of nsP4; and nsP4 itself is a short-lived protein degraded by the ubiquitin-dependent N-end rule pathway.
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PMID:Sindbis virus RNA polymerase is degraded by the N-end rule pathway. 192 57

Mini-F plasmids cannot replicate in Escherichia coli strains (delta rpoH) lacking sigma 32, presumably because transcription of the repE gene encoding the replication initiator protein (RepE protein) depends mostly on RNA polymerase containing sigma 32. We have isolated and characterized mini-F mutants able to replicate in delta rpoH cells. Contrary to the initial expectation, five mutants with mutations in the repE coding region that produce altered RepE proteins were obtained. The mutations caused replacement of a single amino acid: the 92nd glutamic acid was replaced by lysine (repE10, repE16, and repE25) or glycine (repE22) or the 109th glutamic acid was replaced by lysine (repE26). These plasmids overproduced RepE protein and exhibited very high copy numbers. Two major activities of mutated RepE proteins have been determined in vivo; the autogenous repressor activity was significantly reduced, whereas the initiator activity was much enhanced in all mutants. These results indicate the importance of a small central region of RepE protein for both initiator and repressor activities. Thus the decreased repE transcription in delta rpoH cells can be compensated for by an increased initiator activity and a decreased repressor activity of RepE, resulting in the increased synthesis of hyperactive RepE protein.
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PMID:Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein. 199 8

The substitution of the evolutionarily conserved Glu-813 for lysine in the beta subunit of RNA polymerase (RNAP) causes a partial loss of function in the assembled RNAP. In the presence of the four ribonucleoside triphosphates, the mutant RNAP displayed a decreased frequency of promoter clearance and diminished elongation rate. Both defects could be compensated by raising the ribonucleoside triphosphate concentration. In the abortive initiation reaction limited by the incomplete set of ribonucleoside triphosphates, the mutant RNAP generated aberrant patterns of products indicative of their enhanced loss from the RNAP-promoter complex. A model is proposed, attributing the multiple effect of the mutation to the malfunctioning of the RNAP active center.
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PMID:A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase. 206 78

A bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the VH protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-2-sulfonyl (Dns) group. This system has been applied to the expression of the VH genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F. Studier) utilize a T7 expression cassette whose activity is initiated by infection with a lambda phage derivative carrying the T7 RNA polymerase gene. The VH proteins were extracted from the bacterial pellet in 8 M urea and purified by chromatography in 8 M urea. Recombinants with the homologous light (L) chains were prepared to yield VHL molecules. These were used to measure intrinsic affinity for Dns-lysine by resonance energy transfer. The association constants were 7 x 10(6) M-1 and 7 x 10(9) M-1 for the low- and high-affinity systems, respectively. These values are not significantly different from those observed with monoclonal antibodies secreted from the corresponding cell lines. This system lends itself to the quantitative evaluation of the binding properties of the VH protein itself as well as the modulation of affinity by site-directed mutagenesis.
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PMID:Bacterial expression of immunoglobulin VH proteins. 210 93


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