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. Author manuscript; available in PMC: 2016 Apr 27.
Published in final edited form as: Cell Rep. 2014 Oct 9;9(2):476–483. doi: 10.1016/j.celrep.2014.09.008

Fig. 2. PPP at the active site of ValS is required for efficient tRNA charging.

Fig. 2

(A) Location of PPP in the active site of ValS relative to Val-AMP (PDB1GAX) (Fukai et al., 2000). (B) Active site of IleS with Ile-AMP (1JZQ) (Nakama et al., 2001). (C) ValS from (A) but with superimposed position of Ile-AMP from (B). (D) as in (C) but with in silico Pro41Gly mutation. (E) Charging efficiency of tRNAVal with valine by wildtype (wt) ValS and ValS mutants as a function of time (min). (F,G) Autoradiograph of TLC separation of (F) γ[32P]-Pi from γ[32P]-ATP, and (G) α[32P]-AMP and α[32P]-ADP from α[32P]-ATP, when wildtype ValS (wt), or ValS mutant(s) were incubated with valine, in the absence (-) and presence (+) of pyrophosphatase (PPi-ase) and/or deacylated tRNAVal. ATP at the origin indicates where the samples were loaded onto the TLC plate. In (F), the migration position of Pi is determined by treatment of γ[32P]-ATP with Apyrase (A), and in (G), the migration of AMP and ADP was determined by treatment of α[32P]-ATP with increasing concentrations of Apyrase.