Figure 1.
The purified Gcd10p/Gcd14p complex has tRNA(m1A)MTase activity specific for gcd10Δ tRNA. Affinity-purified FlagGcd10p/Gcd14p complexes were resolved on a Superose-6 FPLC column precalibrated with commercial standards of 4–670 kDa (Bio-Rad). The elution positions and masses of the standards are shown on top above the line. Aliquots containing equal portions of every other column fraction in the range predetermined to contain the complexes were separated by using SDS/PAGE followed by immunoblot analysis with antibodies against Gcd10p or Gcd14p (A) or by silver staining (B). Input lane contains an aliquot of the material loaded on the column. (C) Aliquots (3%) of the indicated fractions were assayed for (m1A)MTase activity in reactions (0.05 ml) containing total tRNA (8.0 μM) isolated from yJA146 (gcd10Δ hcIMT4) or yJA158 (GCD10 hcIMT4) and S-adenosyl-l-[methyl-14C]methionine (10 μM). The acid-insoluble portion of each reaction was collected on nitrocellulose filters and dried, and the radioactivity was determined by liquid scintillation. The radioactivity incorporated is plotted against fraction number.