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Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification

N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1,...

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Autores principales: Perrochia, Ludovic, Guetta, Dorian, Hecker, Arnaud, Forterre, Patrick, Basta, Tamara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
RNA
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814370/
https://www.ncbi.nlm.nih.gov/pubmed/23945934
http://dx.doi.org/10.1093/nar/gkt720
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author Perrochia, Ludovic
Guetta, Dorian
Hecker, Arnaud
Forterre, Patrick
Basta, Tamara
author_facet Perrochia, Ludovic
Guetta, Dorian
Hecker, Arnaud
Forterre, Patrick
Basta, Tamara
author_sort Perrochia, Ludovic
collection PubMed
description N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.
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spelling pubmed-38143702013-11-04 Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification Perrochia, Ludovic Guetta, Dorian Hecker, Arnaud Forterre, Patrick Basta, Tamara Nucleic Acids Res RNA N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation. Oxford University Press 2013-11 2013-08-14 /pmc/articles/PMC3814370/ /pubmed/23945934 http://dx.doi.org/10.1093/nar/gkt720 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle RNA
Perrochia, Ludovic
Guetta, Dorian
Hecker, Arnaud
Forterre, Patrick
Basta, Tamara
Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification
title Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification
title_full Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification
title_fullStr Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification
title_full_unstemmed Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification
title_short Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t(6)A tRNA modification
title_sort functional assignment of keops/ekc complex subunits in the biosynthesis of the universal t(6)a trna modification
topic RNA
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814370/
https://www.ncbi.nlm.nih.gov/pubmed/23945934
http://dx.doi.org/10.1093/nar/gkt720
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