Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification |
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Authors: | Ludovic Perrochia Dorian Guetta Arnaud Hecker Patrick Forterre Tamara Basta |
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Affiliation: | 1.Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and 2.Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France |
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Abstract: | N6-threonylcarbamoyladenosine (t6A) 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 t6A 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 t6A. 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 t6A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation. |
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