Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site
Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site
复制标题
DOI：
10.1073/pnas.1219305110
复制
发表时间：
2013-04-23
影响因子：
11.1
通讯作者：
Moore, Melissa J.
中科院分区：
文献类型：
--
作者：
Crawford, Daniel J.;Hoskins, Aaron A.;Moore, Melissa J.
从信使RNA （pre- mrna）的前体中去除内含子需要剪接体将内含子末端紧密结合，但这种结合何时以及如何发生尚不清楚。我们利用单分子荧光共振能量转移和共定位单分子光谱研究了内含子末端聚集在一起的过程，这两种方法的结合可以直接揭示大分子机器中的构象转变如何与特定的组装和拆卸事件相耦合。FRET测量表明，在剪接体组装过程中，5'剪接位点和分支位点保持物理分离，只有在剪接体被激活催化后才会相互靠近，此时前mrna变得高度动态。在剪接途径的后期分离化学位点对于防止在不正确的位点剪接可能是至关重要的。
Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.