前体mRNA中存在的非编码内含子序列需要在翻译前去除,它们通过剪接体切除,由许多蛋白质和RNA成分组成的多兆分子机器。DEAH盒ATP酶Prp2在前mRNA剪接过程中起着至关重要的作用,因为它确保了剪接体的催化激活。尽管与其他剪接体DEAH-box解旋酶具有很高的结构相似性,Prp2似乎不作为RNA解旋酶,而是作为RNA依赖性核糖核蛋白颗粒修饰ATP酶。剪接体DEAH盒ATPasesPrp43和Prp22以及相关RNA解旋酶MLE的最新晶体结构,与RNA复合有助于更好地理解RNA结合和持续合成能力如何在这个解旋酶家族中实现。为了阐明Prp2功能的不同方式,在ADP-BeF3-和poly-U12RNA的存在下,使嗜热ChaetomiumPrp2的N末端截短构建体结晶。与Prp43的ADP-BeF3-和RNA结合结构相比,精制结构揭示了解旋酶核心的几乎相同的构象,并且C末端结构域仅发生了微小的变化。然而,Prp2和Prp43在螺旋束域的钩环和环中不同,它与钩环相互作用,并在3\'堆叠后立即唤起不同的RNA构象。在用Prp2序列替换Prp43中的这些环残基时,取消了Prp43的展开活动。此外,可以在其中一个Prp2结构中鉴定出ATP水解后γ-磷酸盐的推定出口隧道。
Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase
Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases,
Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of
Prp2, an N-terminally truncated construct of Chaetomium thermophilum
Prp2 was crystallized in the presence of ADP-BeF3- and a poly-U12 RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF3-- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However,
Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3\' stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.