Fig. 7: The ATPase activity of Bud32 is potentiated by tRNA binding.
From: A substrate binding model for the KEOPS tRNA modifying complex
a–f ATPase activity analysis of the indicated archaea KEOPS proteins in the presence and absence of mjtRNALysUUU. Activity was monitored using the ADP Glo assay. Displayed results represent the average luminescence (n = 3 independent experiment samples, ±SD) for each reaction condition. c In addition to mjtRNALysUUU, the effect the alternate KEOPS substrates mjtRNAMetCAU, mjtRNAAsnGUU, mjtRNAThrGGU, and the KEOPS non-substrates mjtRNAAspGUC, mjtRNACysGCA mjtRNAValCAC, mjtRNAArgGCG, and mjtRNAAlaGGC on ATPase activity were also examined. d In addition to wild-type mjtRNALysUUU, the effect of mjtRNALysUUUΔCCA on ATPase activity was also examined. e The mutant KEOPS proteins denoted by Mut (in red) are mjCgi121Met60Glu, mjBud32Arg60Glu, mjKae1Arg163Asp and pfPcc1Arg63Asp. g A model for the KEOPS t6A catalytic cycle. Cgi121 can bind tRNA independent of other KEOPS subunits (yellow box). Binding of the Cgi121–tRNA complex to the Bud32–Kae1–Pcc1 complex delivers the tRNA substrate to KEOPS (blue circle). Binding of tRNA to KEOPS (and minimally the Cg121–Bud32–Kae1 subcomplex) activates the ATPase activity of Bud32 which in turns activates the t6A modification activity of Kae1. Dissociation of Cgi121 and t6A-modified tRNA from KEOPS completes a catalytic cycle.
