Fig. 5: All KEOPS subunits contribute to a binding surface for tRNA substrate.
From: A substrate binding model for the KEOPS tRNA modifying complex
a Ribbon representation of the mjCgi121–mjBud32–mjKae1 complex bound to modeled tRNA. Residues in close proximity to the tRNA-binding surface of Bud32 that were selected for mutational analysis are shown in stick representation. The Pcc1 subunit was omitted for clarity. b Analysis of tRNA binding by mjCgi121–mjBud32 subcomplexes using a filter binding assay. mjCgi121 was tested alone or in complex with mjBud32 wild-type or the indicated mutants. c Competitive displacement of a 647-CCA probe from binding to the wild-type mjCgi121–mjBud32 complex or a complex containing the indicated Bud32 mutants by increasing concentration of mjtRNALys. Displacement of the 647-CCA probe was monitored by fluorescence polarization (FP) (n = 3 independent experiment samples, ±SD). d In vitro t6A modification activity analysis of reconstituted arKEOPS complexes with wild-type mjBud32 and the indicated Bud32 mutants. Representative HPLC profiles of nucleoside composition for each reaction (left) and quantification (right) showing the average t6A content normalized to the content of uridine (n = 3 independent experiment samples, ±SD). e Ribbon representation of the mjCgi121–mjBud32–mjKae1–pfPcc1 complex bound to modeled tRNA. Residues in close proximity to the tRNA-binding surface on Kae1 and Pcc1 that were selected for mutational analysis are shown in stick representation. mjCgi121 and mjBud32 were omitted for clarity. f In vitro t6A modification activity analysis of reconstituted arKEOPS complexes with WT or the indicated Pcc1 and Kae1 mutants. Representative HPLC profiles of nucleoside composition for each reaction (top) and quantification (bottom) showing the average t6A content normalized to the content of uridine (n = 3 independent experiment samples, ±SD).
