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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments.
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PMID:The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans. 2468 30

The influenza RNA-dependent RNA polymerase is a core enzyme required for both transcription and replication of the virus RNA genome, making it a potential drug target for the influenza virus. To detect the feature of cap-dependent transcription of influenza B virus (FluB) polymerase, we determined the crystal structures of the wild-type FluB polymerase PB2 subunit cap-binding domain (PB2cap) with bound GDP and the mutant FluB Q325F PB2cap with bound m(7)GDP or GDP. These structures revealed that, distinct from influenza A virus (FluA) PB2cap, the guanine and ribose moieties of substrates invert in FluB PB2caps. Moreover, we characterized the substrate specificity and affinity of the PB2caps using isothermal titration calorimetry. FluB PB2cap has a weaker affinity for m(7)GDP than FluA PB2cap. Unlike FluA PB2cap that has a preference for m(7)GDP in comparison with GDP, FluB PB2cap shows an analogous affinity for both substrates. Replacement of FluB PB2 Glu(325) by Phe, the corresponding residue of FluA PB2, increased the binding affinity of FluB PB2cap for m(7)GDP to a level approximate to that of FluA PB2cap and caused a significant higher affinity to GDP. This study indicated that FluB PB2cap has a unique cap recognition mechanism compared with FluA PB2cap, providing molecular insight into inhibitor design targeting FluB PB2cap.
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PMID:The crystal structure of the PB2 cap-binding domain of influenza B virus reveals a novel cap recognition mechanism. 2569 68

In vivo incorporation of a phenylalanine (Phe) analogue, p-azidophenylalanine (p-N₃Phe) into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket, in which the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) was expressed under the control of T7 promoters. In this study, biosynthesized aECM-CS5-ELF containing p-N₃Phe (aECM-CS5-ELF-N₃) was coupled with alkyne-containing vinyl polymers prepared via controlled radical polymerization of three vinyl monomers, (styrene, acrylamide, and N-isopropylacrylamide) using a trithiocarbonate as the RAFT agent. Grafting of the vinyl polymers onto the aECM was accomplished via a copper-catalyzed alkyne-azide click reaction. The lower critical transition temperature (LCST) was evaluated, as well as the solubility in aqueous and organic media, which are dependent on the incorporation ratio of p-N₃Phe and species of graft chains, in which the LCST behavior was altered remarkably when poly(N-isopropylacrylamide) moieties were attached as side chains. Circular dichroism measurements indicate conformational change was not induced by the grafting. Specific adhesion of human umbilical vein endothelial cells (HUVECs) onto the aECM-CS5-ELF-N₃-graft-poly(N-isopropylacrylamide) composite surface and subsequent temperature-sensitive detachment were also demonstrated.
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PMID:Click Grafting of Alkyne-containing Vinyl Polymers onto Biosynthesized Extracellular Matrix Protein Containing Azide Functionality and Adhesion Control of Human Umbilical Vein Endothelial Cells. 2629 60

Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg(2+) ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe(364) and Pro(357), which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe(364) to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.
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PMID:Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo. 2713 34

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3D^pol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3D^pol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A₂₄ Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237F^HF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237I^LF) and W237L^LF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3D^pol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3D^pol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237F^HF substitution or W237I^LF and W237L^LF mutations were highly attenuated in animals. Our study shows that obtaining 3D^pol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.
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PMID:Attenuation of Foot-and-Mouth Disease Virus by Engineered Viral Polymerase Fidelity. 2851 97


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