Fig. 7: Functional analysis of unique contacts between the anticodon loop of tRNA and Kae1.
From: Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32
A Cryo-EM density in surface representation and fitted atomic model in cartoon representation highlighting contacts between the anticodon stem of the tRNA in its distorted conformation with a structured Kae1 loop 3 (residues 23–43) and Pcc1 α-helix 1 (residues 62–66). B, C Weblogo sequences showing (B) the conservation of Gly38 and Pro41 in Kae1 orthologs and (C) the conservation of Arg63 in Pcc1 orthologs. D Cryo-EM density in surface representation and fitted atomic model in cartoon representation highlighting contacts between the anticodon stem of the tRNA in its native-like conformation and Kae1-specific insert 1 (residues 156–163). E Weblogo logo sequences showing the conservation of the Kae1-specific-insert residues contacting tRNA. F Competitive displacement of an Alexa-647 labeled CCA-tail probe (647-CCA) from KEOPS WT or KEOPS reconstituted with the indicated Kae1 mutants by titration of tRNALys. Displacement of the 647-CCA probe was monitored by fluorescence polarization (FP). Respective mean IC50 values for the displacement are shown (n = 3 technical replicates, error bars represent ± standard deviation SD). G ATPase activity analysis of KEOPS reconstituted with Kae1 WT or the indicated mutants in the presence and absence of tRNALys. Activity was monitored using the ADP Glo assay. Displayed results center represent the mean luminescence for each reaction condition, error bars represent ±SD (n = 3 technical replicates). H t6A modification activity analysis of KEOPS reconstituted with Kae1 WT or the indicated mutants. tRNALys was used as substrate. Representative HPLC profiles of nucleoside composition for each reaction are shown at left. Quantification shows center of mean t6A content normalized to the content of uridine and error bars represent ±SD (n = 3 technical replicates). Source data are provided as a Source Data file.
