Figure 1. (a) Schematic diagram of the MS2–MBP protein. In the protein used in these studies, the loop responsible for dimerization [ 16] has been replaced by the indicated amino acids. Another variant of the MS2–MBP protein that is extensively used [ 15] has two mutations in the loop (V75E and A81G) that prevent dimerization [ 16]; (b) The three consecutive MS2 hairpins [ 17] that are used to attach the MS2–MBP fusion protein as a molecular affinity tag for the pre‐mRNA substrate in splicing. Suitable restriction sites used by Zhou et al. [ 17] to selectively include an increasing number of hairpins at the 3′ end are indicated. Three hairpins were found to be optimal for spliceosome purification, without showing any interference with splicing; (c) Schematic diagram of the different pre‐mRNA substrates used. The filled boxes represent the exons, and thick lines represent the introns. The three MS2 hairpin loops are shown schematically.

Figure 2. Size fractionation of the C complex by gradient sedimentation. The C complex was assembled on MS2–MBP‐tagged PM5 pre‐mRNA for 3 h in a 12 ml splicing reaction. RNase H was then activated by adding an excess of M6 and M12 oligonucleotides (as described in the text). The mixture was fractionated on a 10–30% (v/v) glycerol gradient in a TST41.14 rotor for 12 h at 25 000 rpm (111 000 × g) at 4 °C. The gradient was fractionated manually into 0.5 ml fractions. (a) A plot of the radioactivity determined in each fraction of one of the six gradients. The material peaking in fractions 17–21, corresponding to the C complex, was selected by affinity to an amylose resin, washed on the matrix, and then eluted with 10 mM maltose in buffer G (as detailed in the text); (b) Recentrifugation of the purified C‐complex on a second, equivalent gradient. This second centrifugation was in the TH660 rotor for 1:47 h at 60 000 rpm (484 300 ×g). The radioactivity of each fraction is plotted. The positions of the E. coli 30S and 50S ribosomal subunits are marked at the top.

Figure 3. Assay of purity of the C‐complex preparation after amylose affinity selection. The RNA composition revealed the presence of U2, U5, and U6 snRNA, the free exon 1, and the exon 2 lariat structure (silver stain). The latter two are identified by comparing the autoradiograph (PI, PhosphorImager scan) with the silver‐stained gel. These data demonstrate the high purity of the C‐complex preparation. A typical Coomassie blue‐stained SDS–polyacrylamide gel (4–12% NuPAGE Novex Bis‐Tris Mini gels, run in MOPS buffer; Invitrogen) is shown at the right.