Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system

Abstract

The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t6A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t6A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t6A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms.

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

“…The other two, TsaC and TsaD, are members of universal families and are associated with the t 6 A pathway in several organisms. The biosynthesis of t 6 A 37 has been reconstituted in vitro in the bacterial species E. coli and Bacillus subtilis (17) and the eukaryotic and archaeal species Saccharomyces cerevisiae and Pyrococcus abyssi, respectively (18,19). The biosynthesis in S. cerevisiae and P. abyssi requires L-threonine, ATP, and CO 2 /HCO 3 Ϫ , analogous to bacteria, but five proteins are necessary as follows: Sua5 (the TsaC homolog), Kae1 (the TsaD homolog), Bud32, Pcc1, and Cgi121 (18).…”

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

“…Extensive structural studies delineate the Kae1 active site and binding surfaces with its partners Pcc1 and Bud32 (Hecker et al 2008;Mao et al 2008;Wan et al 2013;Zhang et al 2015). Interestingly, only one of our many point mutations directly affects any of these functionally characterized regions ( Fig.…”

“…Indeed, subcellular redirection of Qri7 to the cytoplasm rescued the slow growth phenotypes of all KEOPS members that are defective for t6A accumulation (Wan et al 2013). Moreover, in vitro synthesis of t6A-modified tRNA was reconstituted solely with purified Sua5, Qri7, and substrate tRNA, in the presence of threonine, bicarbonate, and ATP (Wan et al 2013). Thus, Kae1/Qri7 is the central downstream enzyme in the generation of t6A-modified tRNA.…”

“…The fact that no other KEOPS components localize to mitochondria led to a suspicion that Qri7 represents a primal core factor that does not require additional presumably accessory KEOPS components. Indeed, subcellular redirection of Qri7 to the cytoplasm rescued the slow growth phenotypes of all KEOPS members that are defective for t6A accumulation (Wan et al 2013). Moreover, in vitro synthesis of t6A-modified tRNA was reconstituted solely with purified Sua5, Qri7, and substrate tRNA, in the presence of threonine, bicarbonate, and ATP (Wan et al 2013).…”

Kinase-associated Endopeptidase 1 (Kae1) Participates in an Atypical Ribosome-associated Complex in the Apicoplast of Plasmodium falciparum

“…The DEZ complex (YgjD-YjeEYeaZ) is the prokaryotic counterpart to the EKC/KEOPS com-plex, and it is made up of Kae1, YrdC, and two proteins of unknown function called YeaZ and YjeE (9,14). Interestingly, it was recently reported in yeast that the mitochondrial Kae1 homologue Qri7 can modify tRNA in the absence of protein binding partners (13).…”