FIG. 7.

Yju2 and a heat-resistant factor(s) are required for the first catalytic reaction after the Prp2 step. (A) Splicing was carried out in Yju2-depleted extracts and reaction mixtures (lane 1) precipitated with the anti-Ntc20 antibody (lanes 3 to 8). Precipitates were incubated in the presence of Mg²⁺ at 25°C for 20 min alone (lane 3) or with the addition of recombinant His-Yju2 (lanes 5 and 6, 50 ng) or affinity-purified Yju2-HA (lanes 7 and 8) from 150 μl yeast extracts and either in combination with 4 μl heat-treated extracts prepared from Yju2-depleted extracts (lanes 4, 6, and 8) or alone (lanes 3, 5, and 7). (B) Splicing was carried out in Yju2-depleted heat-inactivated prp2-1 extracts, followed by addition of glucose (final concentration, 5 mM) to deplete ATP. Affinity-purified V5-tagged Prp2 was then added and incubated for 10 min, and the Prp2-bound spliceosome was precipitated with the anti-V5 antibody. Affinity-purified Yju2-HA and/or heat-treated extracts were then added to precipitates for incubation in the presence (lanes 7 to 10) or absence (lanes 3 to 6) of 2 mM ATP at 25°C for 20 min. ΔE, heat-treated extracts; RXN, reaction; PAS, protein A-Sepharose.