tRNA binding to KEOPS human protein complex

N6-theronylcarbomyl adenosine (t6A) is a universal tRNA posttranscriptional modification that promotes translation fidelity and is therefore required for the fitness of virtually all living cells. Kae1, the t6A modifying enzyme in eukaryotes and archea, resides within the KEOPS complex with three auxiliary subunits of unknown function namely Cgi121, Bud32 and Pcc1. Through biochemical and X-ray crystallographic analyses we show that Cgi121 functions to recruit substrate tRNA to KEOPS via its universal 3’-CCA tail. Incorporation of the Cgi121-tRNA structure into a composite model of KEOPS reveals an extended tRNA contact surface that surprisingly spans all four subunits. Comprehensive mutational and functional analyses validate the relevance of the extended tRNA-binding surface in vivo and in vivo. Together this work provides insight into the t6A catalytic cycle and its regulation through the cooperative action of all four KEOPS subunit through direct contacts with tRNA.