BINDING OF THE CBP2 PROTEIN TO A YEAST MITOCHONDRIAL GROUP-I INTRON REQUIRES THE CATALYTIC CORE OF THE RNA
BINDING OF THE CBP2 PROTEIN TO A YEAST MITOCHONDRIAL GROUP-I INTRON REQUIRES THE CATALYTIC CORE OF THE RNA
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DOI：
10.1101/gad.5.10.1870
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发表时间：
1991-10-01
影响因子：
10.5
通讯作者：
CECH, TR
中科院分区：
文献类型：
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作者：
GAMPEL, A;CECH, TR
酵母CBP 2基因产物是体内线粒体细胞色素B前体mRNA末端内含子（b15）剪接所必需的。 在体外，b15 RNA仅在高MgCl 2浓度（50 mM）下有效自我剪接;在5 mM MgCl 2下，有效剪接需要纯化的CBP 2蛋白。 为了确定由蛋白质识别的b15内的序列，我们构建了RNA的缺失和取代突变体。 通过它们抑制野生型b15 RNA的蛋白依赖性剪接的能力来评估它们与CBP 2的结合。 几个区域，包括大的L1和L 8环，可以被删除而不影响结合。 因此，可以将它们排除在作为关键承认要素的考虑之外。 相反，其他变化阻止RNA结合CBP 2，也损害了自我剪接。 因此，要么催化核心直接接触蛋白质，要么核心的完整性是正确展示结合蛋白质的其他RNA序列所必需的。 结果与其中CBP 2蛋白通过结合并稳定RNA的活性结构来促进剪接的模型一致。 然而，提出了一种更具体的模型，其中蛋白质特异性地增强催化所需的Mg 2+结合。
The yeast CBP2 gene product is required for the splicing of the terminal intron (b15) of the mitochondrial cytochrome b pre-mRNA in vivo. In vitro, b15 RNA self-splices efficiently only at high MgCl2 concentrations (50 mM); at 5 mM MgCl2, efficient splicing requires purified CBP2 protein. To determine the sequences within b15 recognized by the protein, we have constructed deletion and substitution mutants of the RNA. Their binding to CBP2 was assessed by their ability to inhibit protein-dependent splicing of the wild-type b15 RNA. Several regions, including the large L1 and L8 loops, can be deleted without affecting binding. They can therefore be eliminated from consideration as critical recognition elements. In contrast, other changes prevent the RNA from binding CBP2 and also impair self-splicing. Thus, either the catalytic core contacts the protein directly, or the integrity of the core is required for proper display of other RNA sequences that bind the protein. The results are consistent with a model in which the CBP2 protein facilitates splicing by binding to and stabilizing the active structure of the RNA. However, a more specific model is proposed in which the protein specifically enhances Mg2+ binding required for catalysis.