(A) Proposed secondary structure of the M. thermautotrophicus (MT) tRNAGln(CUG) transcript. (B) Representative time-course for formation of Glu-tRNAGln by MT GluRSND. Ap indicates the position where the nonaminoacylated 3′-terminal monophosphorylated A76 nucleotide migrates. Glu-Ap indicates the position of migration for the glutamylated A76 nucleotide. (C) Glutamylation of MT tRNAGln(CUG). Open symbols represent reactions in which the tRNA substrate was prefolded by heating to 80°C, followed by addition of Mg2+ to 10 mM final concentration and slow cooling to 21°C. These glutamylation reactions were performed at 21°C (open circles), 37°C (open inverted triangles), or 45°C (open squares). The solid circles represent a reaction in which the tRNA substrate was heated to 80°C, followed by addition of Mg2+ to a 10 mM final concentration, rapid cooling to 0°C, and subsequent glutamylation performed at 21°C. The curve depicted by solid diamonds represents the aminoacylation time-course at 21°C for a tRNA sample that was subjected to the rapid-cooling protocol and then subsequently incubated for 300 min at 37°C.
