Folding of the MT tRNAGln in vitro transcript. (A) Experimental design for the coupled folding and aminoacylation assay. See text for details. (B) Reaction progress curves for aminoacylation at 21°C, using tRNA substrate taken from a folding reaction performed at 21°C. Reactions were initiated by addition of GluRSND to a final concentration of 100 nM, after folding incubation times (t1) of 2.75 min (triangles), 20.5 min (squares), and 185.2 min (diamonds). The ordinate here and in panels C through F is normalized such that the 65% aminoacylation plateau represents the maximum fraction of Glu-tRNAGln that can be synthesized. (C) Reaction progress curves for aminoacylation at 21°C, using tRNA substrate taken from a folding reaction performed at 0°C. Reactions were initiated by addition of GluRSND to a final concentration of 1 μM, after folding incubation times (t1) of 0.58 min (triangles), 11.12 min (squares), 16.37 min (diamonds), and 120 min (inverted triangles). (D) Replots of aminoacylation burst amplitude with folding time t1. Solid-colored symbols are derived from the burst amplitude data in panel B for folding at 21°C, and open symbols are derived from the burst amplitude data in panel C for folding at 0°C. Solid black circles represent additional data points not shown in panel B. (E) Replots of aminoacylation burst amplitude with folding time t1 for folding reactions performed at 21°C at GluRSND concentrations of 100 nM (filled symbols; these data are the same as shown in panel D) and 1 μM (open circles). (F) Reaction progress curves at 21°C showing effects of tRNAGln preincubation for 10 min in the presence of 100 nM GluRSND (open circles), and preincubation without GluRSND (open squares). Inverted triangles depict the reaction progress curve at 21°C for an experiment in which pyrophosphatase was added together with GluRSND when initiating the reaction.
