Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing.
Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing.
复制标题
偏见的布朗棘轮会导致前MRNA重塑并在第一步剪接之前捕获。
DOI：
10.1038/nsmb.2704
复制
发表时间：
2013-12
影响因子：
16.8
通讯作者：
Walter NG
中科院分区：
文献类型：
--
作者：
Krishnan R;Blanco MR;Kahlscheuer ML;Abelson J;Guthrie C;Walter NG
剪接体是一种动态的核糖核蛋白（RNP）机器，在真核生物前体mRNA剪接的两个酯交换步骤中催化内含子的去除。在这里，我们使用单分子荧光共振能量转移来监测亲和纯化的剪接体中前体mRNA的5′剪接位点（5′SS）和分支点（BP）的距离，该剪接体在第一个剪接步骤之前被DExD/H盒解旋酶Prp 2中的突变所阻止。添加重组Prp 2与NTP和蛋白辅因子Spp 2一起重排剪接体-底物复合物，以可逆地探索与近端5′SS和BP的构象，以适应化学。Cwc 25的加入则强烈地使这种平衡偏向于近端构象，促进有效的第一步剪接。因此，剪接体作为一个偏置的布朗棘轮机的功能，其中解旋酶解锁热波动，随后由辅因子“棘爪”，一个原则可能普遍存在于许多解旋酶驱动的RNP整流。
The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns in the two transesterification steps of eukaryotic pre-mRNA splicing. Here we used single molecule fluorescence resonance energy transfer to monitor the distance of the 5′ splice site (5′SS) and branchpoint (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately prior to the first splicing step. Addition of recombinant Prp2 together with NTP and protein cofactor Spp2 rearranges the spliceosome-substrate complex to reversibly explore conformations with proximal 5′SS and BP that accommodate chemistry. Addition of Cwc25 then strongly biases this equilibrium towards the proximal conformation, promoting efficient first-step splicing. The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor “pawl”, a principle possibly widespread among the many helicase-driven RNPs.