Madhani H D, Guthrie C
Department of Biochemistry and Biophysics, University of California at San Francisco 94143-0448.
Genes Dev. 1994 May 1;8(9):1071-86. doi: 10.1101/gad.8.9.1071.
Putative components of the spliceosomal active site include a bulged helix between U2 and U6 snRNAs (U2-U6 helix I) and the adjacent ACAGAG hexanucleotide in U6. We have developed an in vivo, bimolecular randomization-selection method to functionally dissect these elements. Although a portion of U2-U6 helix I resembles the G-binding site of group I introns, the data are inconsistent with an analogous functional role for this structure in the spliceosome. Instead, analysis of several novel covariants supports the existence of a structure in which the helix I bulge engages in a tertiary interaction with the terminal residue of the U6 hexanucleotide (ACAGAG). Such a higher order structure, together with other known interactions, would juxtapose the two clusters of residues of the U2-U6 complex that are specifically required for the second chemical step of pre-mRNA splicing with the 3' splice site. Indeed, mutations in the residues that participate in the tertiary interaction affect both the efficiency and fidelity of 3' splice site usage.
剪接体活性位点的推定组分包括U2和U6小核RNA之间的一个凸起螺旋（U2-U6螺旋I）以及U6中相邻的ACAGAG六核苷酸。我们开发了一种体内双分子随机化选择方法来从功能上剖析这些元件。尽管U2-U6螺旋I的一部分类似于I组内含子的G结合位点，但数据并不支持该结构在剪接体中具有类似的功能作用。相反，对几个新的共变体的分析支持了一种结构的存在，其中螺旋I凸起与U6六核苷酸（ACAGAG）的末端残基进行三级相互作用。这样一种高阶结构，连同其他已知的相互作用，会使U2-U6复合体中两个特定参与前体mRNA剪接第二步化学反应的残基簇与3'剪接位点并列。实际上，参与三级相互作用的残基发生突变会影响3'剪接位点使用的效率和保真度。
