Bao Penghui, Wu Qi-Jia, Yin Ping, Jiang Yanfei, Wang Xu, Xie Mao-Hua, Sun Tao, Huang Lin, Mo Ding-Ding, Zhang Yi
Department of Biochemistry and Molecular Biology, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Nucleic Acids Res. 2008 Dec;36(21):6934-43. doi: 10.1093/nar/gkn824. Epub 2008 Oct 31.
Self-splicing of group I introns is accomplished by two sequential ester-transfer reactions mediated by sequential binding of two different guanosine ligands, but it is yet unclear how the binding is coordinated at a single G-binding site. Using a three-piece trans-splicing system derived from the Candida intron, we studied the effect of the prior GTP binding on the later omegaG binding by assaying the ribozyme activity in the second reaction. We showed that adding GTP simultaneously with and prior to the esterified omegaG in a substrate strongly accelerated the second reaction, suggesting that the early binding of GTP facilitates the subsequent binding of omegaG. GTP-mediated facilitation requires C2 amino and C6 carbonyl groups on the Watson-Crick edge of the base but not the phosphate or sugar groups, suggesting that the base triple interactions between GTP and the binding site are important for the subsequent omegaG binding. Strikingly, GTP binding loosens a few local structures of the ribozyme including that adjacent to the base triple, providing structural basis for a rapid exchange of omegaG for bound GTP.
I 组内含子的自我剪接是通过由两种不同鸟苷配体的顺序结合介导的两个连续的酯转移反应完成的，但尚不清楚这种结合是如何在单个 G 结合位点上进行协调的。使用源自念珠菌内含子的三片段反式剪接系统，我们通过检测第二个反应中的核酶活性，研究了先前 GTP 结合对随后 ωG 结合的影响。我们发现，在底物中与酯化的 ωG 同时或在其之前添加 GTP 会强烈加速第二个反应，这表明 GTP 的早期结合促进了随后 ωG 的结合。GTP 介导的促进作用需要碱基沃森-克里克边缘上的 C2 氨基和 C6 羰基，而不是磷酸或糖基团，这表明 GTP 与结合位点之间的碱基三联体相互作用对于随后 ωG 的结合很重要。引人注目的是，GTP 结合会使核酶的一些局部结构松弛，包括与碱基三联体相邻的结构，为 ωG 与结合的 GTP 的快速交换提供了结构基础。
