Figure 1. An ATP-dependent activity rejects pre-mRNA reversibly prior to 5′ splice site cleavage, if the rate of 5′ splice site cleavage is slow.

(A) An ATP-dependent activity competes with 5′ splice site cleavage. U6/sU80 (Sp) spliceosomes stalled in Mg²⁺ on an ACT1 pre-mRNA substrate were affinity-purified and then either frozen (no incubation) or incubated with the indicated metals and with or without ATP-Mg²⁺. The migration of the splicing species is indicated on the right. The bottom panel shows the efficiency of 5′ splice site cleavage. Values are represented as means ± standard error of mean (SEM) from three experiments. (See also Figure S1).

(B) ATP-dependent rejection is reversible. Spliceosomes were stalled and purified as in A and then either frozen (no incubation) or incubated −/+ ATP. Spliceosomes incubated with ATP were then washed to remove ATP and incubated further −/+ ATP. Dashed lines indicate that samples were not incubated further.

(C) In the absence of ATP, U6/sU80 (Sp) spliceosomes catalyzed 5′ splice site cleavage in Mg²⁺ but with an apparent rate 10-fold lower than that in Mn²⁺ and 20 fold lower than that in Cd²⁺ (k_Mg = 0.010 ± 0.006 min⁻¹; k_Mn = 0.13 ± 0.02 min⁻¹; k_Cd = 0.22 ± 0.02 min⁻¹ – errors reflect the error in the fit of the data points (see Experimental Procedures)). Reactions were as in A but without ATP. Error bars represent ± SEM of each data point for three experiments.