Isolation of Ltp26/Ltp28 100-kD complex from L. tarentolae mitochondria. (A) The model mRNA editing substrate contains four 4-thiouridines (in bold) in the first editing site. The anchor region is labeled, and the locations of the original U nucleotides which were mutated to non-U nucleotides outside the editing site are indicated by boxes. In five cases, the complementary nucleotides were also mutated to preserve base pairing. (B) The annealed labeled mRNA-gRNA substrate was incubated with mitochondrial extract and UV-irradiated for 0–20 min. The extract was fractionated on a 12% SDS gel and the gel was exposed to a Phosphoimager plate. RNA-protein crosslinks I and II are indicated. (C) Crosslink I in the S100 mitochondrial extract (lane 1) was followed through ammonium sulfate precipitation (lane 2), heparin affinity chromatography (lane 3), and Superose 12 size-fractionation (lane 4; see Materials and Methods). Active fractions were analyzed on a 12% SDS gel and stained with Coomassie Blue.
