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Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity

In archaeal rRNAs, the isomerization of uridine into pseudouridine (Ψ) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Ψ-synthase activity is the aCBF5–aNOP10 protein pair. The crystal structure of the aCBF5–aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1...

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Autores principales: Manival, Xavier, Charron, Christophe, Fourmann, Jean-Baptiste, Godard, François, Charpentier, Bruno, Branlant, Christiane
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1361308/
https://www.ncbi.nlm.nih.gov/pubmed/16456033
http://dx.doi.org/10.1093/nar/gkj482
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author Manival, Xavier
Charron, Christophe
Fourmann, Jean-Baptiste
Godard, François
Charpentier, Bruno
Branlant, Christiane
author_facet Manival, Xavier
Charron, Christophe
Fourmann, Jean-Baptiste
Godard, François
Charpentier, Bruno
Branlant, Christiane
author_sort Manival, Xavier
collection PubMed
description In archaeal rRNAs, the isomerization of uridine into pseudouridine (Ψ) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Ψ-synthase activity is the aCBF5–aNOP10 protein pair. The crystal structure of the aCBF5–aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1 Å resolution. In this structure, protein aNOP10 has an extended shape, with a zinc-binding motif at the N-terminus and an α-helix at the C-terminus. Both motifs contact the aCBF5 catalytic domain. Although less efficiently as does the full-length aNOP10, the aNOP10 C-terminal domain binds aCBF5 and stimulates the RNA-guided activity. We show that the C-terminal domain of aCBF5 (the PUA domain), which is wrapped by an N-terminal extension of aCBF5, plays a crucial role for aCBF5 binding to the guide sRNA. Addition of this domain in trans partially complement particles assembled with an aCBF5ΔPUA truncated protein. In the crystal structure, the aCBF5–aNOP10 complex forms two kinds of heterotetramers with parallel and perpendicular orientations of the aNOP10 terminal α-helices, respectively. By gel filtration assay, we showed that aNOP10 can dimerize in solution. As both residues Y41 and L48 were needed for dimerization, the dimerization likely takes place by interaction of parallel α-helices.
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spelling pubmed-13613082006-02-08 Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity Manival, Xavier Charron, Christophe Fourmann, Jean-Baptiste Godard, François Charpentier, Bruno Branlant, Christiane Nucleic Acids Res Article In archaeal rRNAs, the isomerization of uridine into pseudouridine (Ψ) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Ψ-synthase activity is the aCBF5–aNOP10 protein pair. The crystal structure of the aCBF5–aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1 Å resolution. In this structure, protein aNOP10 has an extended shape, with a zinc-binding motif at the N-terminus and an α-helix at the C-terminus. Both motifs contact the aCBF5 catalytic domain. Although less efficiently as does the full-length aNOP10, the aNOP10 C-terminal domain binds aCBF5 and stimulates the RNA-guided activity. We show that the C-terminal domain of aCBF5 (the PUA domain), which is wrapped by an N-terminal extension of aCBF5, plays a crucial role for aCBF5 binding to the guide sRNA. Addition of this domain in trans partially complement particles assembled with an aCBF5ΔPUA truncated protein. In the crystal structure, the aCBF5–aNOP10 complex forms two kinds of heterotetramers with parallel and perpendicular orientations of the aNOP10 terminal α-helices, respectively. By gel filtration assay, we showed that aNOP10 can dimerize in solution. As both residues Y41 and L48 were needed for dimerization, the dimerization likely takes place by interaction of parallel α-helices. Oxford University Press 2006 2006-02-02 /pmc/articles/PMC1361308/ /pubmed/16456033 http://dx.doi.org/10.1093/nar/gkj482 Text en © The Author 2006. Published by Oxford University Press. All rights reserved
spellingShingle Article
Manival, Xavier
Charron, Christophe
Fourmann, Jean-Baptiste
Godard, François
Charpentier, Bruno
Branlant, Christiane
Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity
title Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity
title_full Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity
title_fullStr Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity
title_full_unstemmed Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity
title_short Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5–aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity
title_sort crystal structure determination and site-directed mutagenesis of the pyrococcus abyssi acbf5–anop10 complex reveal crucial roles of the c-terminal domains of both proteins in h/aca srnp activity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1361308/
https://www.ncbi.nlm.nih.gov/pubmed/16456033
http://dx.doi.org/10.1093/nar/gkj482
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