Figure 4

Chymotryptic protection in the RNA binding domain of SRP72. a. Digestion of purified 72 j (lane 1) with Chymotrypsin for 3, 6 or 10 minutes (lanes 2 to 4). Similarly, digestion of purified 72 k (lane 5) for 3, 6 or 10 minutes (lanes 6 to 8). Lane 9, the 72 l fragment (lacking residues 584 to 590) treated with Chymotrypsin for 10 minutes; lane 10, the 585/6 polypeptide, generated by a 10 minute chymotryptic digest of 72 k, retained on Ni-NTA magnetic agarose beads (see Methods for details). All samples were analyzed by electrophoresis on 12.5 percent SDS polyacrylamide gels followed by staining of the polypeptides with Coomassie blue. Molecular mass markers in kDa are indicated on the left and the migrations of Chymotrypsin (CT) and the his-tagged C-terminal 585/6 fragment are marked on the right. b. Digestion of 1.2 μg 72 j with Chymotrypsin in the absence of RNA (lane 1) or in the presence of increasing amounts of human Δ35 RNA (lanes 2 to 4)[23]. Indicated are molecular mass markers and the migrations of Chymotrypsin (CT), the 72 j polypeptide, and the his-tagged oligopeptide generated by the cleavages of the Tyrosines 585 or 586. c. Binding of human Δ35 to increasing amounts of 72 j. d. Results from gelshift experiment for the binding data shown in Figure 5c. Indicated are the mobilities of the free Δ35 RNA (fr) and its complex with 72 j (cp). Lane numbers correspond to the seven data points shown in Figure 5c. e. Similar to Figure 5 d, showing the formation of a complex between the 72 j and the 5e SRP RNA.