Figure 4
In vitro self-splicing of B.c .I4 and B.th .I6a wild-type (WT) and mutant constructs in KCl-containing buffer. (A) B.c.I4 deleted of the entire 3' extension (B.c.I4_dS1S2); (B) B.c.I4 WT; (C) B.c.I4 deleted of the entire 3' extension and the branchsite adenosine (B.c.I4_dA_dS1S2); (D) B.c.I4 deleted of the branchsite adenosine only (B.c.I4_dA); (E) B.th.I6a WT; and (F) B.th.I6a deleted of the entire 3' extension (B.th.I6a_dS1S2). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM KCl, and 100 mM MgCl2 at 47°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. The various splicing products are labeled on the sides. The weak bands corresponding to the linear forms of B.th.I6a (panels E and F) are marked by arrowheads and were identified by size. "dS1S2" and "dA" refer to deletion of the entire 3' extension or the branchsite adenosine, respectively