Figure 3.
Identification of the missing nondiscriminating ERS and of the AdT tRNAQ substrate. (A) Aminoacylation curves of unfractionated mtRNA with mERS and subsequent addition of mERS (-○-) or cERS (-◆-) when the aminoacylation plateau with mERS was reached (16 min, dashed line). Autoradiogram of the TLC plate analyzing transamidation by purified yeast GatFAB AdT of [14C]E-mtRNA (40 μL) generated at plateau level by mERS (inset i) or generated at plateau level by combined charging with mERS and cERS (inset ii). (B) Identification by Northern blotting of the tRNAQ and tRNAE species present in our unfractionated mtRNA preparations. 15 μg of unfractionated mtRNA (mi) was hybridized with probes specific for mitochondrial-encoded tRNAQ (mtRQ), tRNAE (mtRE), and nuclear-encoded tRNAQ (ctRQ) and tRNAK (tRK1) shown previously to be imported into mitochondria. As a control, 15 μg of unfractionated ctRNA (cy) was probed with the same probes. (C) Charging curves of purified mtRNAQ and mtRNAE by cERS (respectively, -◆- and -●-) and mERS (respectively, -◇- and -○-). (Inset) Transamidation of the pure mischarged [14C]E-mtRNAQ by pure yeast GatFAB AdT.