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)

A genomic clone of the small, round-structured virus Southampton virus (SV) was constructed from a set of overlapping PCR amplicons. Sequence analysis confirmed the absence of mutations and accurate ligation of the PCR products. The SV cDNA was cloned into a vector for in vitro production of RNA and subsequent translation by rabbit reticulocyte lysate. Two polypeptides corresponding to the N-terminal and C-terminal regions of the viral polyprotein were expressed in Escherichia coli and used to produce murine antisera for detection of translation products. Three major translation products of 113, 48, and 41 kDa were identified in a coupled transcription-translation system. The large 113-kDa protein reacted with antisera raised against the C-terminal region of the polyprotein and represents a precursor of the viral RNA polymerase. The 48-kDa protein detected in vitro reacted specifically with antisera raised against the polyprotein N terminus, showing that translation was initiated in SV at the three tandem in-frame AUG codons at the 5' end of the genome. A series of nested 3' deletions of the large open reading frame encoding the viral polyprotein was used to define the translation initiation site and genomic location of the viral protease. The results are consistent with a model in which translation of the viral genome is initiated at one of the three in-frame AUG codons starting at nucleotide position 5 and in which active viral protease is produced following translation of a region located between NheI (nucleotide 3052) and SphI (nucleotide 4056), resulting in rapid cleavage of a large precursor protein. Abolition of the viral 3C-like protease activity by site-directed mutagenesis of the putative active-site cysteine (Cys-1238) resulted in production of a large protein of approximately 200 kDa which reacted with both N-terminal and C-terminal antisera. Two potential polyprotein cleavage sites containing the preferred picornaviral QG recognition site were identified on either side of the putative 2C-like helicase region of the polyprotein. Proteolysis at these positions would give rise to products with relative molecular masses identical to those of the products detected in the rabbit reticulocyte system. Site-directed mutagenesis was used to introduce a single base change which resulted in the substitution of glutamine residues with proline residues at amino acids 399 and 762. These mutations completely abolished cleavage of the polyprotein at these positions and gave rise to alternative products with molecular masses which matched the predicted sizes for a single cleavage at either Q-399 or Q-762. These data indicate that the small, round-structured virus Southampton virus produces a 3C-like protease which has two primary cleavage sites at positions 399 and 762. Proteolytic cleavage at these positions releases the putative viral 2C-like helicase.
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PMID:Polyprotein processing in Southampton virus: identification of 3C-like protease cleavage sites by in vitro mutagenesis. 864 93

Bioactive peptide hormones and neurotransmitters are required for neuroendocrine regulation of cellular functions. Importantly, proteolytic processing of inactive neuropeptide precursors is required to generate physiologically active peptide hormones and neurotransmitters. Studies of the processing enzymes require authentic neuropeptide precursors as substrates, rather than peptide substrates. This study demonstrates an efficient method to general 35S-precursors from cloned cDNAs by in vitro transcription and translation. In vitro transcription of neuropeptide cDNAs with SP6 RNA polymerase generates large amounts (micrograms) of corresponding RNAs. Subsequent in vitro translation of RNAs with wheat germ extract and 35S-methionine generates large quantities of 35S-precursors (10-25 million cpm 35S-precursor protein per reaction) with high specific radioactivity. The radiolabeled precursor substrates offer a reliable, sensitive and accurate method for detecting the proteolytic activity. Importantly, specific detection of the primary proenkephalin processing activity in chromaffin granules by 35S-enkephalin precursor as substrate, but not by peptide methylcoumarinamide (MCA) substrates, illustrates the significance of using full-length precursor to detect appropriate processing enzymes. This study demonstrates that efficient production of radiolabeled neuropeptide precursors by in vitro transcription and translation will be useful for in vitro assays of relevant processing proteases.
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PMID:Production of radiolabeled neuropeptide precursors by in vitro transcription and translation. 891 65

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
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PMID:Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2). 902 6

The sigma (sigma) subunit of prokaryotic RNA polymerase is required for specific recognition of promoter DNA sequences and transcription initiation. Regulation of gene expression can therefore be achieved by modulating the activity of the sigma subunit. In Bacillus subtilis the mother cell-specific sporulation sigma factor, sigmaK, is synthesized as a precursor protein, pro-sigmaK, with a 20-amino acid pro sequence. This pro sequence renders sigmaK inactive for directing transcription of sigmaK-dependent genes in vivo until the pro sequence is proteolytically removed. To understand the role of the pro sequence in controlling sigmaK activity, we have constructed NH2-terminal truncations of pro-sigmaK and characterized their behavior in vitro at the gerE promoter. In this report we show that the pro sequence inactivates sigmaK by interfering with the ability of sigmaK to associate with the core subunits of polymerase and also influences the interactions between holoenzyme and promoter DNA. Additionally, removal of as few as 6 amino acids (pro-sigmaKDelta6) is sufficient to activate pro-sigmaK for DNA binding and transcription initiation. Surprisingly, pro-sigmaKDelta6 binds to DNA with higher affinity and stimulates transcription 30-fold more efficiently than sigmaK, under certain conditions.
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PMID:The role of the pro sequence of Bacillus subtilis sigmaK in controlling activity in transcription initiation. 938 52

We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.
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PMID:Generation of infectious pancreatic necrosis virus from cloned cDNA. 976 36

This paper presents evidence that Thosea asigna virus (TaV) has a unique capsid expression strategy and is a member of the Nudaurelia beta-like genus of the Tetraviridae. Electron microscopy of TaV particles indicated a 38 nm, T = 4 icosahedral capsid similar in structure to that of Nudaurelia beta virus (NbetaV). TaV particles have a buoyant density of 1.296 g/cm3 in CsCl and consist of two capsid proteins of 56 and 6 kDa. The virus genome contains a genomic RNA molecule of 6.5 kb and a subgenomic molecule of 2.5 kb. Northern blotting of TaV RNA indicated a genomic organization similar to that of NbetaV. The capsid gene of TaV is carried on both the genomic and subgenomic RNA molecules, while the RNA polymerase gene is present only on the genomic RNA. Cloning and sequencing of the TaV capsid gene identified an open reading frame that could potentially encode a capsid precursor protein of up to 82.5 kDa. The N-terminal sequences of the capsid proteins were compared with the nucleotide sequence of the capsid open reading frame. The sequences indicate that the pre-protein is cleaved at two positions to produce the 56 and 6 kDa capsid proteins as well as a predicted third protein that was not detected in the mature virion. Phylogenetic analysis of the capsid proteins indicated that TaV is more closely related to NbetaV than to the Nudaurelia omega-like viruses. The eight beta-sheets that make up a jelly roll structure in the TaV capsid protein were identified by computer analysis.
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PMID:A novel capsid expression strategy for Thosea asigna virus (Tetraviridae). 1042 56

Feline calicivirus (FCV) capsid protein was expressed in feline cells employing the vaccinia virus MVA/T7 RNA polymerase system. The precursor protein was processed to a mature size protein that assembled to virus like particles (VLPs).
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PMID:Feline calicivirus capsid protein expression and self-assembly in cultured feline cells. 1051 71

The interaction of the human adenovirus proteinase (AVP) with various DNAs was characterized. AVP requires two cofactors for maximal activity, the 11-amino acid residue peptide from the C-terminus of adenovirus precursor protein pVI (pVIc) and the viral DNA. DNA binding was monitored by changes in enzyme activity or by fluorescence anisotropy. The equilibrium dissociation constants for the binding of AVP and AVP-pVIc complexes to 12-mer double-stranded (ds) DNA were 63 and 2.9 nM, respectively. DNA binding was not sequence specific; the stoichiometry of binding was proportional to the length of the DNA. Three molecules of the AVP-pVIc complex bound to 18-mer dsDNA and six molecules to 36-mer dsDNA. When AVP-pVIc complexes bound to 12-mer dsDNA, two sodium ions were displaced from the DNA. A Delta of -4.6 kcal for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy results from nonspecific interactions between the AVP-pVIc complex and DNA. The cofactors altered the interaction of the enzyme with the fluorogenic substrate (Leu-Arg-Gly-Gly-NH)2-rhodamine. In the absence of any cofactor, the Km was 94.8 microM and the kcat was 0.002 s(-1). In the presence of adenovirus DNA, the Km decreased 10-fold and the kcat increased 11-fold. In the presence of pVIc, the Km decreased 10-fold and the kcat increased 118-fold. With both cofactors present, the kcat/Km ratio increased 34000-fold, compared to that with AVP alone. Binding to DNA was coincident with stimulation of proteinase activity by DNA. Although other proteinases have been shown to bind to DNA, stimulation of proteinase activity by DNA is unprecedented. A model is presented suggesting that AVP moves along the viral DNA looking for precursor protein cleavage sites much like RNA polymerase moves along DNA looking for a promoter.
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PMID:Human adenovirus proteinase: DNA binding and stimulation of proteinase activity by DNA. 1168 32

Poliovirus (PV) VPg is a genome-linked protein that is essential for the initiation of viral RNA replication. It has been well established that RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine (Y3) in VPg by the viral RNA polymerase 3D(pol), but it is not yet known whether the substrate for uridylylation in vivo is the free peptide itself or one of its precursors. The aim of this study was to use complementation analyses to obtain information about the true in vivo substrate for uridylylation by 3D(pol). Previously, it was shown that a VPg mutant, in which tyrosine 3 and threonine 4 were replaced by phenylalanine and alanine (3F4A), respectively, was nonviable. We have now tested whether wild-type forms of proteins 3B, 3BC, 3BCD, 3AB, 3ABC, and P3 provided either in trans or in cis could rescue the replication defect of the VPg(3F4A) mutations in the PV polyprotein. Our results showed that proteins 3B, 3BC, 3BCD, and P3 were unable to complement the RNA replication defect in dicistronic PV or dicistronic luciferase replicons in vivo. However, cotranslation of the P3 precursor protein allowed rescue of RNA replication of the VPg(3F4A) mutant in an in vitro cell-free translation-RNA replication system, but only poor complementation was observed when 3BC, 3AB, 3BCD, or 3ABC proteins were cotranslated in the same assay. Interestingly, only protein 3AB but not 3B and 3BC, when provided in cis by insertion of a wild-type 3AB coding sequence between the P2 and P3 domains of the polyprotein, supported the replication of the mutated genome in vivo. Elimination of cleavage between 3A and 3B in the complementing 3AB protein, however, led to a complete lack of RNA replication. Our results suggest that (i) VPg has to be delivered to the replication complex in the form of a large protein precursor (P3) to be fully functional in replication; (ii) the replication complex formed during PV replication in vivo is essentially inaccessible to proteins provided in trans, even if the complementing protein is translated from a different cistron of the same RNA genome; (iii) 3AB is the most likely precursor of VPg; and (iv) Y3 of VPg has an essential function in RNA replication in the context of both VPg and 3AB.
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PMID:Tyrosine 3 of poliovirus terminal peptide VPg(3B) has an essential function in RNA replication in the context of its precursor protein, 3AB. 1736 Jul 46

Transcription factor IIA (TFIIA) is one of the general transcription factors for RNA polymerase II and composed of three subunits, TFIIAalpha, TFIIAbeta and TFIIAgamma. TFIIAalpha and TFIIAbeta are encoded by a single gene (TFIIAalphabeta) and mature through internal cleavage of TFIIAalphabeta. In this study, we found that structures of TFIIAalphabeta and TFIIAgamma are highly homologous with each mammalian counterpart. Exon-intron organizations of the human and chicken TFIIA genes were also homologous. The sequence of the cleavage region of the chicken TFIIAalphabeta precursor protein was fitted to the consensus cleavage recognition site. It was thus demonstrated that TFIIA is conserved in vertebrates. TFIIA proteins are present ubiquitously in chicken tissues. Fluorescent in situ hybridization revealed that TFIIAalphabeta and TFIIAgamma genes are located in chromosome 5 and a mini-chromosome, respectively. We generated semi-knockout chicken DT40 cells for TFIIAalphabeta and TFIIAgamma genes with high homologous recombination efficiencies, whereas we failed to establish double-knockout cells for each gene. It is thought that both genes for TFIIA are required in vertebrates. TFIIA siRNA resulted in deceleration of cell growth rate, suggesting that, consistent with those of knockout assays, TFIIA is associated with cell growth regulation.
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PMID:Chromosomal position, structure, expression, and requirement of genes for chicken transcription factor IIA. 1754 29


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