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

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.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions

“…Consistent with this model is the observation that the B. subtilis homologs of TsaBDE are capable of t 6 A 37 biosynthesis in the presence of TC-AMP and tRNA and the absence of YwlC, the TsaC homolog (17). The argument is strengthened by the ability of P. abyssi Kae1, the TsaD homolog, to bind TC-AMP and catalyze the transfer to tRNA (26). However, several studies have shown that E. coli TsaC selectively binds hypomodified tRNA (24,25,27), indicating TsaC may have a more substantial, diverse role in bacteria than in eukaryotes because S. cerevisiae Sua5 does not exhibit RNA binding activity (18).…”

Protein folding and tRNA biology

“…The t6A modification requires several enzymatic steps (Perrochia et al 2013;Thiaville et al 2014). In Archaea and Eukarya, this process involves a universal protein (Sua5) and the complex KEOPS/EKC (Srinivasan et al 2011).…”

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

“…In addition, considering the particular limiting nature of the initiator tRNA for cell and animal growth (5,7) and as this tRNA is modified by t 6 A, we were prompted to investigate the impact of t 6 A in whole animal context using Drosophila. All previous studies on t 6 A function and synthesis have been performed in unicellular organisms or cells in culture (15)(16)(17)(18)(19)(20)(21). In this study, we address for the first time the role of t 6 A in a metazoan.…”

“…It stabilizes codon-anticodon interaction (13), suggesting that t 6 A might have a role regulating protein synthesis initiation. The modification was discovered over 40 years ago (14), but the enzymes involved in t 6 A synthesis were only identified and characterized in all domains of life in the last few years (15)(16)(17)(18)(19)(20)(21) and recently renamed (22). In yeast, components of the threonyl-carbamoyl transferase complex (TCTC, previously named KEOPS/EKC (kinase, endopeptidase and other proteins of small size/endopeptidase-like and kinase associated to transcribed chromatin) are required for t 6 A synthesis.…”