Abstract：
Structural rearrangement of the activated spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) spliceosome. In the absence of Spp2, the B(act) spliceosome efficiently triggers Prp2\'s NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the spliceosome. Transformation of the B(act) to the B* spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of spliceosomes, Spp2 enhances Prp2\'s RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2\'s ATPase activity to remodeling of the spliceosome into a catalytically active machine.
摘要：
激活的剪接体（B（act））的结构重排以产生催化活性复合物（B*）是由DEAH-boxNTPasePrp2与G-patch蛋白Spp2合作介导的。然而,目前尚不清楚Prp2水解ATP的能量如何与机械功耦合，以及Spp2在这一过程中起什么作用。使用纯化的剪接系统，我们证明，在B（act）剪接体中，不需要将Prp2招募到其真正的结合位点。在没有Spp2的情况下，B（act）剪接体有效地触发Prp2的NTPase活性，但是NTP水解不与核糖核蛋白（RNP）重排偶联，从而导致剪接体的催化活化。B（act）向B*剪接体的转化仅在Spp2存在时发生，并伴随着Prp2的解离及其NTPase活性的降低。在没有剪接体的情况下,Spp2增强Prp2的RNA依赖性ATP酶活性而不影响其RNA亲和力。我们的数据表明，Spp2在将Prp2的ATPase活性与剪接体重塑为催化活性机器中起着重要作用。
