Wang Yanli, Juranek Stefan, Li Haitao, Sheng Gang, Tuschl Thomas, Patel Dinshaw J
Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA.
Nature. 2008 Dec 18;456(7224):921-6. doi: 10.1038/nature07666.
Here we report on a 3.0 A crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2'-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.
在此，我们报道了野生型嗜热栖热菌（Thermus thermophilus）的Argonaute与5'-磷酸化的21核苷酸引导DNA以及在第10 - 11步含有防止切割错配的20核苷酸靶RNA形成的三元复合物的3.0埃晶体结构。种子片段（第2至8位）采用A - 螺旋样的沃森 - 克里克配对双链体，引导链的两端锚定在复合物中。在二元复合物中插入引导链第10和11位碱基之间的精氨酸，使复合物锁定在无活性构象，在三元复合物形成时释放。在形成更开放的三元复合物时，Argonaute的含PAZ和PIWI结构域的叶之间的核酸结合通道变宽。通过测定含有位置依赖性碱基错配、凸起和2'-O-甲基修饰的三元复合物的切割活性，建立了结构与功能的关系。与三元复合物的几何结构一致，位于靶标而非引导链种子片段中的凸起更易被容纳，并且它们的复合物具有催化活性。
