Abelson John, Hadjivassiliou Haralambos, Guthrie Christine
Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA.
Methods Enzymol. 2010;472:31-40. doi: 10.1016/S0076-6879(10)72017-6.
The spliceosome is a complex small nuclear (sn)RNA-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. For each splicing event, the spliceosome is assembled de novo on a pre-mRNA substrate and a complex series of assembly steps leads to the active conformation. To comprehensively monitor pre-mRNA conformational dynamics during spliceosome assembly, we developed a strategy for single-molecule FRET (smFRET) that utilizes a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. In this chapter, we describe the identification of Ubc4 pre-mRNA that is efficiently spliced in vitro and the methods we have developed for the chemical synthesis of fluorescent Ubc4 pre-mRNA for smFRET.
剪接体是一种复杂的小核（sn）RNA-蛋白质机器，它通过两个连续的磷酸转移反应从前体mRNA中去除内含子。对于每一次剪接事件，剪接体在一个前体mRNA底物上从头组装，一系列复杂的组装步骤导致活性构象的形成。为了全面监测剪接体组装过程中前体mRNA的构象动态变化，我们开发了一种单分子荧光共振能量转移（smFRET）策略，该策略利用一种小的、能高效剪接的酵母前体mRNA，即Ubc4，其中供体和受体荧光团分别置于与5'和3'剪接位点相邻的外显子中。在本章中，我们描述了在体外能高效剪接的Ubc4前体mRNA的鉴定，以及我们为化学合成用于smFRET的荧光Ubc4前体mRNA所开发的方法。
