Fig. 8: Functional analysis of unique contacts between the D-arm motif of tRNA and the conserved tail of Bud32.
From: Structures of KEOPS bound to tRNA reveal functional roles of the kinase Bud32
A Cryo-EM density volume and atomic model of the distorted tRNA highlighting the interaction of the flipped base of G26 with the side chain of Arg530 in the C-terminal tail of Bud32. B Weblogo logo sequences showing conservation of Arg530 in Bud32 orthologs. C Weblogo logo sequences showing conservation of G26 in KEOPS substrate tRNAs and tRNAAlaGCG orthologs (top), in tRNALysUUU orthologs (middle), and in non-KEOPS substate tRNAs (bottom). D Competitive displacement of an Alexa-647 labeled CCA-tail probe (647-CCA) from KEOPS reconstituted with Bud32 WT or the indicated mutants by titration of tRNALys. KDead corresponds to a Bud32 D451A active site mutation that generally disables phospho-transfer activity in the eukaryotic protein kinase superfamily. 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 ± SD). E ATPase activity analysis of KEOPS reconstituted with Bud32 WT or the indicated Bud32 mutants in the presence or 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). F t6A modification activity analysis of KEOPS reconstituted with Bud32 WT or the indicated mutants on tRNALys WT or the G26U mutant. Representative HPLC profiles of nucleoside composition for each reaction are shown at right. Quantification shows center of mean t6A content normalized to the content of uridine and error bars represent ±SD (n = 3 technical replicates). G t6A modification activity analysis of KEOPS reconstituted with Bud32 WT or the indicated mutants on tRNALys WT as a function of enzyme concentration. Quantification shows center of mean t6A content normalized to the content of uridine and error bars represent ±SD (n = 3 technical replicates). H t6A modification activity analysis of KEOPS reconstituted with Bud32 WT or the indicated mutants on tRNALys WT or the tRNALys G26U mutant as a function of time. 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.
