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)

We report the identification of three new alpha-amanitin resistance mutations in the gene encoding the largest subunit of mouse RNA polymerase II (RPII215). These mutations are clustered in a region of the largest subunit that is important for transcription elongation. This same domain has been identified as the site of alpha-amanitin resistance mutations in both Drosophila and Caenarhabditis elegans. The sequences encompassing this cluster of mutations are highly conserved among RNA polymerase II genes from a number of species, including those that are naturally more resistant to alpha-amanitin suggesting that this region of the largest subunit is critical for a conserved catalytic function. The mutations reported here change leucine 745 to phenylalanine, arginine 749 to proline, or isoleucine 779 to phenylalanine. Together with the previously reported asparagine 792 to aspartate substitution these mutations define a potential alpha-amanitin binding pocket in a region of the mouse subunit that could be involved in translocation of polymerase during elongation.
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PMID:Clustered alpha-amanitin resistance mutations in mouse. 789 49

Genetic and molecular analysis in Drosophila melanogaster identifies eight suppressor mutations in the second largest subunit of RNA polymerase II. The suppressor mutations fall into two classes: five are strong, result from the same serine to cysteine amino acid residue substitution and rescue one conditional lethal allele in the largest subunit of RNA polymerase II; three are mild, result from a change in the same methionine residue to either isoleucine or valine, are located seven amino acid residues away from the strong suppressors and rescue two conditional lethal alleles in the largest subunit. Sequence analysis of the three regions around these mutations demonstrates that they are located within highly conserved domains but fails to explain the observed genetic interactions. One of the conditional lethal alleles maps within a region previously reported to share sequence similarity to Escherichia coli DNA polymerase I. As the gross structure of RNA polymerase II and DNA polymerase I is similar, even though their primary sequence is not, we predict that more similarities exist but may be too highly divergent to be detected by normal homology searches. We identify the most similar regions between each of the three conserved domains of RNA polymerase II, identified as functionally important because of the mutations we isolated, and DNA polymerase I. Molecular modeling these regions of RNA polymerase II onto the tertiary structure of DNA polymerase I predicts that all lie adjacent to the DNA binding cleft in positions such that they could interact with the phosphate backbone of DNA. This juxtaposition of mutations in the two largest subunits of RNA polymerase II suggest a mechanism for their genetic interactions.
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PMID:Molecular modeling of RNA polymerase II mutations onto DNA polymerase I. 796 18

Four amatoxin-binding proteins with KD values in the nanomolar range, 3 monoclonal antibodies and RNA polymerase II, were studied with respect to their affinities to 24 alpha-amanitin derivatives with modified side chains. From KD values we estimated the amounts of binding energy that single side chains of the amatoxins contribute to complex formation. Ile6, previously identified by X-ray analysis to be part of a beta-turn (Kostansek EC, Lipscomb WN, Yocum RR, Thiessen WE, 1978, Biochemistry 17:3790-3795) proved to be of outstanding importance in all complexes. Replacement of the isoleucine with alanine reduced the affinity to all binding proteins to < 1%, suggesting a strong hydrophobic interaction. A strong effect was also seen when Gly5 was replaced with alanine, suggesting that the absence of a side chain in proximity to the beta-turn is likewise important. In addition to the beta-turn, each of the proteins showed at least 2 other points of strong contact formed by hydrogen bonds. Donors are the indole NH of 6'-hydroxy-Trp4 and OH of hydroxy-Pro2 and dihydroxy-Ile3. All the antibodies, but not RNA polymerase II, recognized the indole nucleus of 6'-hydroxy-Trp4. The geometric arrangement of the 4 strongest contact points suggests that the amatoxin binding site is different in each of the 4 proteins, except for the 2 antibodies raised in the same animal. Here, most of the contact points were identical but differed in strength of interaction. The method of structural analysis presented in this study is useful for identifying contact sites in complexes of proteins with peptides of rigid conformation. Furthermore, the method complements X-ray data by providing information on the amount of binding energy contributed by single structural elements.
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PMID:A beta-turn in alpha-amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies. 806 5

Molecular recognition of Escherichia coli tRNA(Ile) by the cognate isoleucyl-tRNA synthetase (IleRS) was studied by analyses of chemical footprinting with N-nitroso-N-ethylurea and aminoacylation kinetics of variant tRNA(Ile) transcripts prepared with bacteriophage T7 RNA polymerase. IleRS binds to the acceptor, dihydrouridine (D), and anticodon stems as well as to the anticodon loop. The "complete set" of determinants for the tRNA(Ile) identity consists of most of the nucleotides in the anticodon loop (G34, A35, U36, t6A37 and A38), the discriminator nucleotide (A73), and the base-pairs in the middle of the anticodon, D and acceptor stems (C29.G41, U12.A23 and C4.G69, respectively). As for the tertiary base-pairs, two are indispensable for the isoleucylation activity, whereas the others are dispensable. Correspondingly, some of the phosphate groups of these dispensable tertiary base-pair residues were shown to be exposed to N-nitroso-N-ethylurea when tRNA(Ile) was bound with IleRS. Furthermore, deletion of the T psi C-arm only slightly impaired the tRNA(Ile) activity. Thus, it is proposed that the recognition by IleRS of all the widely distributed identity determinants is coupled with a global conformational change that involves the loosening of a particular set of tertiary base-pairs of tRNA(Ile).
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PMID:Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli. 811 89

Promoter-specific transcription by silkworm RNA polymerase III is dependent on several transcription factors (TFs) in addition to the polymerase itself. The activities present in silk gland nuclear extracts that are necessary to reconstitute transcription from class III genes in vitro have been resolved into several partially purified components. These include TFIIIR, which is unusual because it is composed of RNA. Here, we identify the RNA that provides TFIIIR activity as silkworm tRNA(IleIAU). This conclusion is based on copurification of tRNA(IleIAU) with TFIIIR activity, TFIIIR activity in synthetic tRNA(Ile), and hybrid selection of TFIIIR activity by antisense tRNA(IleIAU). We have tested the ability of a variety of other tRNAs to stimulate transcription and find that TFIIIR activity is highly specific to silkworm tRNA(IleIAU).
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PMID:TFIIIR is an isoleucine tRNA. 819 4

The antimalarial activity of rifampicin, a specific inhibitor of bacterial ribonucleic acid (RNA) polymerase, was confirmed with Plasmodium falciparum in vitro and with P. chabaudi in vivo. The viability of ring forms of P. falciparum, measured by [3H]hypoxanthine and [14C]isoleucine uptake, was significantly reduced within 5 h of exposure to 2.5 microM rifampicin, the 50% inhibitory concentration. Streptolydigin and tagetitoxin, other specific inhibitors of bacterial RNA polymerase, were much less effective as antimalarials. A rifampicin-tolerant sub-line of P. falciparum was selected in vitro. When released from drug pressure, the tolerant line showed appreciably greater rates of incorporation of precursors and growth than the parent line, but over a period of months these characteristics gradually reverted. Rifampicin was effective against a chloroquine-resistant line of P. falciparum and the rifampicin-tolerant line had increased chloroquine sensitivity. Treatment of patent parasitaemias of P. chabaudi in mice with more than 100 mg/kg rifampicin twice daily significantly reduced the parasitaemia within 24 h and parasites were barely detectable on blood films by the fourth day. Recrudescence occurred on release of drug pressure.
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PMID:Antimalarial activity of rifampicin in vitro and in rodent models. 833 32

Vaccinia virus (vv) mRNA capping enzyme is composed of a large and a small subunit encoded by genes D1 and D12, respectively. A 38-kDa interfering polypeptide is copurified with the vaccinia virus capping enzyme overproduced in Escherichia coli, but the origin of this polypeptide is unknown (P. Guo and B. Moss, 1990, Proc. Natl. Acad. Sci. USA 87, 4023-4027). This polypeptide competes with the large subunit in binding to the small subunit during the assembly of the heterodimeric enzyme in the cell, resulting in a reduced yield of the active enzyme. Results from the studies of ribosome-binding site replacement, frame shifting, DNA deletion, and in vitro mutagenesis showed that the interfering polypeptide originated from a new translation initiation site within the D1 gene. Transfection of a plasmid containing an internal eukaryotic ribosome binding site into monkey kidney cells infected with vv producing T7 RNA polymerase resulted in the expression of the large subunit up to 30% of total cellular radiolabeled protein; however, the 38-kDa polypeptide was not detected. This finding suggests that the initiation site was recognized only by E. coli, not by eukaryotic cells. The Shine-Dalgarno sequence is not found in the corresponding region preceding the putative start codon, indicating that an unusual mechanism for ribosome binding exists. Mutagenesis of the putative initiation codon of the interfering polypeptide from ATG (Met), coding for residue 498 of the large subunit, to ATA (Ile) eliminated the expression of the interfering polypeptide. A stable and active mutant enzyme was expressed in E. coli HMS174(DE3) cell without the presence of the interfering polypeptide.
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PMID:Tracking and elimination of an interfering polypeptide coexpressed with the vaccinia virus mRNA capping enzyme overproduced in Escherichia coli. 838 35

The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction.
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PMID:Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C. 862 28

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.
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PMID:Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit. 862 88

We identified mutations in the gene for nsP2, a nonstructural protein of the alphavirus Sindbis virus, that appear to block the conversion of the initial, short-lived minus-strand replicase complex (RCinitial) into mature, stable forms that are replicase and transcriptase complexes (RCstable), producing 49S genome or 26S mRNA. Base changes at nucleotide (nt) 2166 (G-->A, predicting a change of Glu-163-->Lys), at nt 2502 (G-->A, predicting a change of Val-275-->Ile), and at nt 2926 (C-->U, predicting a change of Leu-416-->Ser) in the nsP2 N domain were responsible for the phenotypes of ts14, ts16, and ts19 members of subgroup 11 (D.L. Sawicki and S.G. Sawicki, Virology 44:20-34, 1985) of the A complementation group of Sindbis virus RNA-negative mutants. Unlike subgroup I mutants, the RCstable formed at 30 degrees C transcribed 26S mRNA normally and did not synthesize minus strands in the absence of protein synthesis after temperature shift. The N-domain substitutions did not inactivate the thiol protease in the C domain of nsP2 and did not stop the proteolytic processing of the polyprotein containing the nonstructural proteins. The distinct phenotypes of subgroup I and 11 A complementation group mutants are evidence that the two domains of nsP2 are essential and functionally distinct. A detailed analysis of ts14 found that its nsPs were synthesized, processed, transported, and assembled at 40 degrees C into complexes with the properties of RCinitial and synthesized minus strands for a short time after shift to 40 degrees C. The block in the pathway to the formation of RCstable occurred after cleavage of the minus-strand replicase P123 or P23 polyprotein into mature nsP1, nsP2, nsP3, and nsP4, indicating that structures resembling RCstable, were formed at 40 degrees C. However, these RCstable or pre-RCstable structures were not capable of recovering activity at 30 degrees C. Therefore, failure to increase the rate of plus-strand synthesis after shift to 40 degrees C appears to result from failure to convert RCinitial to RCstable. We conclude that RCstable is derived from RCinitial by a conversion process and that ts14 is a conversion mutant. From their similar phenotypes, we predict that other nsP2 N-domain mutants are blocked also in the conversion of RCinitial to RCstable. Thus, the N domain of nsP2 plays an essential role in a folding pathway of the nsPs responsible for formation of the initial minus-strand replicase and for its conversion into stable plus-strand RNA-synthesizing enzymes.
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PMID:Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein. 862 44


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