Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.

Ludovic Perrochia (1) , Dorian Guetta , Arnaud Hecker (1) , Patrick Forterre (1) , Tamara Basta (1)

1 IGM - Institut de génétique et microbiologie [Orsay]

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.

Domaines: Biochimie, Biologie Moléculaire; Microbiologie et Parasitologie

Ludovic Perrochia, Dorian Guetta, Arnaud Hecker, Patrick Forterre, Tamara Basta. Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.. Nucleic Acids Research, 2013, 41 (20), pp.9484-99. ⟨10.1093/nar/gkt720⟩. ⟨hal-00881058⟩