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

Nucleic Acids Res. 2013 Nov;41(20):9484-99. doi: 10.1093/nar/gkt720. Epub 2013 Aug 14.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / metabolism
  • Adenosine Monophosphate / analogs & derivatives
  • Adenosine Monophosphate / chemistry
  • Adenosine Triphosphatases / metabolism
  • Allosteric Regulation
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / isolation & purification
  • Archaeal Proteins / metabolism*
  • Catalytic Domain
  • Dimerization
  • Iron / chemistry
  • Iron-Binding Proteins / chemistry
  • Iron-Binding Proteins / metabolism
  • Molecular Docking Simulation
  • Nucleotides / metabolism
  • Protein Subunits / chemistry
  • Protein Subunits / isolation & purification
  • Protein Subunits / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism
  • Pyrococcus abyssi / enzymology
  • RNA, Transfer / chemistry
  • RNA, Transfer / metabolism*

Substances

  • Archaeal Proteins
  • Iron-Binding Proteins
  • Nucleotides
  • Protein Subunits
  • N(6)-(N-threonylcarbonyl)adenosine
  • Adenosine Monophosphate
  • RNA, Transfer
  • Iron
  • Protein-Serine-Threonine Kinases
  • Adenosine Triphosphatases
  • Adenosine