Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

Effects of lethal alanine mutations on branchpoint RNA binding. (A) Aliquots (3 µg) of purified recombinant wild-type (WT) Msl5-(KH-QUA2) and the R172A, R190A, L256A, and L259A mutants were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated at left. (B) RNA binding reaction mixtures containing 25 mM Tris–HCl, pH 7.5, 1 mM DTT, 10% glycerol, 100 nM 5′ ³²P-labeled 11-mer RNA as specified, and either no protein (−) or 500 nM wild-type or mutants Msl5-(KH-QUA2) as specified were incubated at 4°C for 20 min. The mixtures were analyzed by native PAGE. An autoradiograph of the gel is shown.