Group II introns deleted for multiple substructures retain self-splicing activity.
Group II introns deleted for multiple substructures retain self-splicing activity.
复制标题
删除多个子结构的第二组内含子保留了自剪接活性。
DOI：
10.1128/mcb.12.5.1950-1958.1992
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发表时间：
1992
影响因子：
5.3
通讯作者：
Perlman,PS
中科院分区：
文献类型：
--
作者：
Koch,JL;Boulanger,SC;Dib-Hajj,SD;Hebbar,SK;Perlman,PS
第二类内含子可以折叠成具有六个主要亚结构或结构域的高度保守的二级结构。已知结构域1和5在自剪接中起关键作用，而结构域2、3、4和6的作用不太清楚。已经建立了结构域5功能的反式分析，表明结构域5在前体RNA上具有一个根据任何二级结构元素都不能预测的结合位置。在本研究中，酵母线粒体γ中oxI基因内含子5的自剪接域被单独或组合缺失。这些突变的内含子被鉴定为体外自剪接反应，作为定位结构域5结合位点的一种手段。结构域2、3、4或6的单个缺失不会阻止在任一剪接连接处的体外反应，尽管结构域6的缺失在一定程度上降低了3‘剪接位点选择的保真度。即使缺少结构域2、4和6的三重缺失也保留了一些自剪接活性。结构域2、3、4和6的缺失阻止了3‘剪接连接处的反应，但不阻止5’连接处的反应。根据这些结果，我们得出结论：结构域5的结合部位在结构域1内，5‘外显子、结构域1和结构域5(加上短连接序列)的复合体构成了该内含子的重要催化核心。
Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. Atransassay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5γ of thecoxIgene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.