Figure 3.

With U6/sU₈₀(S_P) RNA, only the first of the two transesterification reactions occurs in the presence of Mn²⁺, which is competitively inhibited by Mg²⁺. a, Mn²⁺ rescues the first but not the second step of splicing in U6/sU₈₀(S_P)-reconstituted reaction. Extract was reconstituted with U6 (lanes 1–6), U6/sU₈₀(R_P) (lanes 7–12), or U6/sU₈₀(S^P) (lanes 13–18) and PRP2 in the presence of Mn²⁺ and Mg²⁺. In each set of six reactions, the concentrations (mM) of Mn²⁺/Mg²⁺ were 0/2.5, 2.5/2.5, 1.25/1.25, 1.7/0.8; 2.0/0.5, 2.5/0. b, Mg²⁺ competitively inhibits the Mn²⁺-rescued reaction. Extract was reconstituted in the absence of PRP2 with U6 (lanes 1–9) or U6/sU₈₀(S_P) (lanes 10–18) and with Mg²⁺ (2.5 mM) as the sole divalent metal ion. Splicing was then triggered by the addition of PRP2 with 1mM Mn²⁺ (lanes 2–9 and 11–18) and an increasing concentration (mM) of Mg²⁺ (2.5, 5.0, 7.5, 10, 15, 20, 25, 30). Reactions in lanes 1 and 2 contained 2.5mM of Mg²⁺ and no Mn²⁺ (asterisk). c, The ratio of splicing efficiency between the U6/sU₈₀(S_P)- and U6-reconstituted reactions (S_P/O). Splicing efficiency in each reaction (Fig. 3b) was calculated as the ratio between the products (IVS–E2*, IVS* and E1–E2) and the total (products plus pre-mRNA).