FIGURE 2.
Affinity purification of human, spliceosomal Bact complexes. (A) Glycerol gradient fractionation of splicing complexes formed on the PM5-20 construct. In vitro splicing, followed by RNAse H digestion, was performed as described in the Materials and Methods. Complexes were then separated on a linear 10%–30% glycerol gradient and the 32P-RNA in each fraction was determined by Cherenkov counting. S-values were determined by comparison with a reference gradient containing prokaryotic ribosomal subunits. Gradient profiles for both PM5-10 (not shown) and PM5-20 complexes were identical. (B,C) Composition of affinity-purified complexes. Gradient fractions containing 45S complexes (fractions 17–21) were pooled and subjected to MS2 affinity selection. (B) RNA was recovered, separated by denaturing PAGE, and visualized by autoradiography (lanes 1 and 2) and by silver staining (lanes 3 and 4). The positions of the snRNAs and the PM5-20 and PM5-10 pre-mRNA are indicated on the right. Asterisk, RNase H degradation product. The other minor bands seen in lanes 1 and 2 do not correspond to splicing intermediates, but are likely degradation products. (C) Protein was recovered from the affinity purified Bact complexes (lanes 1 and 2), analyzed by SDS-PAGE on an 8%/14% polyacrylamide step gel, and visualized by staining with Coomasie. The size of molecular weight markers (lane 3) is indicated on the right.