Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 7.5-kb plus-stranded genomic RNA of rabbit hemorrhagic disease virus contains two open reading frames of 7 kb (ORF1) and 351 nucleotides (ORF2) that cover nearly 99% of the genome. The aim of the present study was to identify the proteins encoded in these open reading frames. To this end, a panel of region-specific antisera was generated by immunization of rabbits with bacterially expressed fusion proteins that encompass in total 95% of the ORF1 polyprotein and almost the complete ORF2 polypeptide. The antisera were used to analyze the in vitro translation products of purified virion RNA of rabbit hemorrhagic disease virus. Our studies show that the N-terminal half of the ORF1 polyprotein is proteolytically cleaved to yield three nonstructural proteins of 16, 23, and 37 kDa (p16, p23, and p37, respectively). In addition, a cleavage product of 41 kDa which is composed of VPg and a putative nonstructural protein of approximately 30 kDa was identified. Together with the results of previous studies which identified a trypsin-like cysteine protease (TCP) of 15 kDa, a putative RNA polymerase (pol) of 58 kDa, and the major capsid protein VP60, our data establish the following gene order in ORF1: NH2-p16-p23-p37 (helicase)-p30-VPg-TCP-pol-VP60-COOH. Immunoblot analyses showed that a minor structural protein of 10 kDa is encoded in ORF2. The data provide the first complete genetic map of a calicivirus. The map reveals a remarkable similarity between caliciviruses and picornaviruses with regard to the number and order of the genes that encode the nonstructural proteins.
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PMID:Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies. 889 21

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.
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PMID:The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). 897 Sep 70

The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication. Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase. Both full-length and truncated forms of NS3 have been identified in infected cells. To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid. The polypeptides detected were NS2B/3 (Mr 83000), NS3 (Mr 69000), NS3' (Mr 50000) and NS3" (Mr 19000). The latter polypeptide has not been previously identified. For DEN-2, it has been proposed that NS3' results from cleavage at the site ...R457R / GR460... within an RNA helicase sequence motif of NS3. Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary.
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PMID:Internal proteolysis of the NS3 protein specified by dengue virus 2. 901 55

The reverse gyrase gene rgy from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and sequenced. The gene is 3,642 bp (1,214 amino acids) in length. The deduced amino acid sequence has relatively high similarity to the sequences of the Methanococcus jannaschii reverse gyrase (48% overall identity), the Sulfolobus acidocaldarius reverse gyrase (41% identity), and the Methanopynrus kandleri reverse gyrase (37% identity). The P. furiosus reverse gyrase is a monomeric protein, containing a helicase-like module and a type I topoisomerase module, which resembles the enzyme from S. acidocaldarius more than that from M. kandleri, a heterodimeric protein encoded by two separate genes. The control region of the P. furiosus rgy gene contains a typical archaeal putative box A promoter element which is located at position -26 from the transcription start identified by primer extension experiments. The initiating ATG codon is preceded by a possible prokaryote-type ribosome-binding site. Purified P. furiosus reverse gyrase has a sedimentation coefficient of 6S, suggesting a monomeric structure for the native protein. The enzyme is a single polypeptide with an apparent molecular mass of 120 kDa, in agreement with the gene structure. The sequence of the N terminus of the protein corresponded to the deduced amino acid sequence. Phylogenetic analysis indicates that all known reverse gyrase topoisomerase modules form a subgroup inside subfamily IA of type I DNA topoisomerases (sensu Wang [J. C. Wang, Annu. Rev. Biochem. 65:635-692, 1996]). Our results suggest that the fusion between the topoisomerase and helicase modules of reverse gyrase occurred before the divergence of the two archaeal phyla, Crenoarchaeota and Euryarchaeota.
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PMID:Characterization of the reverse gyrase from the hyperthermophilic archaeon Pyrococcus furiosus. 904 34

Translation of eukaryotic mRNAs is generally initiated by the scanning ribosome mechanism. This can be downregulated by high affinity protein binding to cap-proximal RNA motifs. Translation can also be regulated by short open reading frames within the 5' -untranslated region. A key factor for initiation is elF4F, in which one of the polypeptide chains, elF4G, seems to have a bridging function and binds three other factors at separate sites: elF4E (the cap-binding factor), the helicase elF4A, and elF3, which also interacts with 40S ribosomal subunits. Initiation is regulated by the MAP kinase and rapamycin-sensitive signalling pathways, which control phosphorylation of elF4E and 4E-BP1, a protein which in the dephosphorylated form binds and sequesters elF4E.
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PMID:Translational controls impinging on the 5'-untranslated region and initiation factor proteins. 911 26

After a brief introduction to the function of the mRNA-specific translation factors eIF4A, eIF4B, and EIF4F, this article presents appropriate methodology for the study of the translation factors associated with the activation of mRNA for translation in eukaryotic systems. The purification of eIF4A, eIF4B, and eIF4F from rabbit reticulocyte lysates is given along with a procedure for the purification of hemoglobin mRNA. These purifications provide reagents for the model assays of RNA binding (as retention on nitrocellulose filters) and RNA-dependent ATP hydrolysis. With additional reagents available as RNA transcripts using either T7 or SP6 polymerase, two additional assays are possible, crosslinking to the oxidized cap of the mRNA or the ATP-dependent reaction of RNA unwinding (helicase assay). Finally, there is a description of the most biological assay for the utilization of natural mRNAs, the synthesis of the authentic polypeptide chain, in this instance hemoglobin. Throughout the portion of this article that deals with the biological assays, helpful hints are provided to ensure that the assay works, and suggestions are provided for control experiments to ensure that an artifact is not being studied. It is hoped that this information will facilitate the study of either the regulation of factor activity (for eIF4A, eIF4B, or eIF4F) or the translation efficiency (sometimes regulated) of various mRNAs.
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PMID:Assays for eukaryotic translation factors that bind mRNA. 912 48

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences.
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PMID:The Rep78 gene product of adeno-associated virus (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences. 915 37

Human coronavirus 229E gene expression involves proteolytic processing of the gene 1-encoded polyproteins pp1a and pp1ab. In this study, we have detected a 71-kDa polypeptide in virus-infected cells that is released from pp1ab by the virus-encoded 3C-like proteinase and that has been predicted to contain both metal-binding and helicase domains. The polypeptide encompasses amino acids Ala-4996 to Gln-5592 of pp1ab and exhibits nucleic acid-stimulated ATPase activity when expressed as a fusion protein with the Escherichia coli maltose-binding protein. These data provide the first identification of a coronavirus open reading frame 1b-encoded enzymatic activity.
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PMID:Identification of an ATPase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229E. 918 39

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.
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PMID:The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain. 920 20

Several examples of direct interactions between helicases and topoisomerases have recently been described. The data suggest a possible cooperation between these enzymes in major DNA events such as the progression of a replication fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, and finally recombination, repair, and genomic stability. A first example is the finding of a strong interaction between T antigen and topoisomerase I in mammalian cells, that may trigger unwinding of the parental DNA strands at the replication forks of Simian Virus 40. A second example is the reverse gyrase from thermophilic prokaryotes, composed of a putative helicase domain, and a topoisomerase domain in the same polypeptide. This enzyme may be required to maintain genomic stability at high temperature. A third example is the finding of an interaction between type II topoisomerase and the helicase Sgs1 in yeast. This interaction possibly allows the faithful segregation of newly replicated chromosomes in eukaryotic cells. A fourth example is the interaction between the same helicase Sgs1 and topoisomerase III in yeast, that may control recombination level and genetic stability of repetitive sequences. Recently, in humans, mutations in genes similar to Sgs1 have been found to be responsible for Bloom's and Werner's syndromes. The cooperation between helicases and topoisomerases is likely to be extended to many aspects of DNA mechanisms including chromatin condensation/decondensation.
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PMID:When helicase and topoisomerase meet! 921 20


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