Institute of Molecular Biophysics, 91 Chiefton Way, Florida State University, Tallahasee, Florida 32306, USA.
Nat Struct Mol Biol. 2007 Dec;14(12):1189-95. doi: 10.1038/nsmb1336.
The most complex RNA pseudouridylases are H/ACA ribonucleoprotein particles, which use a guide RNA for substrate capture and four proteins (Cbf5, Nop10, Gar1 and L7Ae/NHP2) for substrate modification. Here we report the three-dimensional structure of a catalytically deficient archaeal enzyme complex (including the guide RNA and three of the four essential proteins) bound to a substrate RNA. Extensive interactions of Cbf5 with one guide-substrate helix and a guide RNA stem shape the forked guide–substrate RNA complex structure and position the substrate in proximity of the Cbf5 catalytic center. Our structural and complementary fluorescence analyses also indicate that precise placement of the target uridine at the active site requires a conformation of the guide–substrate RNA duplex that is brought about by the previously identified concurrent interaction of the guide RNA with L7Ae and a composite Cbf5-Nop10 surface, and further identify a residue that is critical in this process.
最复杂的 RNA 假尿嘧啶核苷转移酶是 H/ACA 核糖核蛋白颗粒，它使用向导 RNA 捕获底物，并使用 4 种蛋白质（Cbf5、Nop10、Gar1 和 L7Ae/NHP2）进行底物修饰。在这里，我们报告了一种催化缺陷的古菌酶复合物（包括向导 RNA 和四种必需蛋白质中的三种）与底物 RNA 结合的三维结构。Cbf5 与一条向导-底物螺旋和一条向导 RNA 茎的广泛相互作用，形成了分叉的向导-底物 RNA 复合物结构，并将底物置于 Cbf5 催化中心的附近。我们的结构和互补荧光分析还表明，将靶尿嘧啶精确地放置在活性位点需要一种向导-底物 RNA 双链的构象，这种构象是由先前鉴定的向导 RNA 与 L7Ae 和复合 Cbf5-Nop10 表面的同时相互作用引起的，并进一步确定了在此过程中起关键作用的一个残基。
