Gampel A, Nishikimi M, Tzagoloff A
Department of Biological Sciences, Columbia University, New York, New York 10027.
Mol Cell Biol. 1989 Dec;9(12):5424-33. doi: 10.1128/mcb.9.12.5424-5433.1989.
The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.
酵母线粒体细胞色素b基因的末端内含子（bI2）是一种I类内含子，在高浓度Mg2+存在的情况下能够在体外进行自我剪接。然而，bI2在体内的切除需要由核基因CBP2编码的一种蛋白质。CBP2蛋白已从野生型酵母线粒体中得到部分纯化，并显示在生理浓度的Mg2+条件下能促进剪接。自我剪接和蛋白质依赖性剪接反应都利用了鸟苷核苷辅因子，这是I类内含子自我剪接反应的标志。此外，消除bI2自催化活性的突变也会阻断蛋白质依赖性剪接。这些结果表明，bI2的蛋白质依赖性切除是一个RNA催化的过程，涉及与I类内含子自我剪接相同的两步转酯机制。我们提出，CBP2蛋白与bI2前体结合，从而稳定RNA的催化活性结构。在没有蛋白质的情况下，高浓度的Mg2+可能会诱导相同或相似的RNA结构。野生型前体可使蛋白质依赖性反应达到饱和，这一观察结果支持了CBP2蛋白与未剪接前体的结合。切除的bI2以及剪接位点附近5'外显子发生突变的剪接缺陷型前体可竞争性抑制蛋白质依赖性剪接，但核心结构发生突变（的剪接缺陷型前体）则不会。CBP2蛋白与前体RNA的结合对5'剪接位点螺旋有影响，这一点可通过抑制外源二核苷酸与内部引导序列的相互作用得到证明。
