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. 2013 Dec 5;42(4):2538–2554. doi: 10.1093/nar/gkt1256

Figure 5.

Figure 5.

Mutational analysis of the RNA helix-destabilizing activity of VP5. (A) Illustration of the mutagenesis strategy. Asterisk indicates sites of replacement with alanine. (B) Expressed and purified MBP-VP5 mutants were subjected to 12% SDS-PAGE followed by Coomassie brilliant blue R250 staining. (C) Helix substrate (0.1 pmol; RNA1/RNA3; upper panel) was reacted with 10 pmol MBP-VP5 wild-type and mutants as indicated (lanes 3–6). Boiled reaction mixture (lane 1) was used as positive control, and the reaction mixture with no protein (lane 2) or supplemented with MBP alone (lane 7) was used as a negative control. (D) Gel mobility shift assays were performed by incubating 0.1 pmol HEX-labeled RNA1 with 10 pmol MBP-VP5 wild type and mutants as indicated (lanes 3–6). Reaction mixture without protein supplementation (lane 1) or with MBP alone (lane 2) was used as a negative control.