Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A 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, 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.
Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France.
Ludovic
L
Perrochia
Dorian
D
Guetta
Arnaud
A
Hecker
Patrick
P
Forterre
Tamara
T
Basta
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000494:Allosteric Regulation; D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D019281:Dimerization; D007501:Iron / Q000737:chemistry; D033862:Iron-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D062105:Molecular Docking Simulation; D009711:Nucleotides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D043903:Pyrococcus abyssi / Q000201:enzymology; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*
D019843:Archaeal Proteins; D033862:Iron-Binding Proteins; D009711:Nucleotides; D021122:Protein Subunits; C003268:N(6)-(N-threonylcarbonyl)adenosine; D000249:Adenosine Monophosphate; D012343:RNA, Transfer; D007501:Iron; D017346:Protein Serine-Threonine Kinases; D000251:Adenosine Triphosphatases; D000241:Adenosine