GROUP-II INTRONS DELETED FOR MULTIPLE SUBSTRUCTURES RETAIN SELF-SPLICING ACTIVITY
GROUP-II INTRONS DELETED FOR MULTIPLE SUBSTRUCTURES RETAIN SELF-SPLICING ACTIVITY
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DOI：
10.1128/mcb.12.5.1950
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发表时间：
1992-05-01
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;PERLMAN, PS
II组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。已经开发了结构域5功能的反式测定，其表明结构域5在前体RNA上具有结合位点，这不是从任何二级结构元件预测的。在这项研究中，酵母线粒体DNA的coxI基因的自剪接组II内含子5-γ被删除的各种内含子结构域，单独和组合。这些突变内含子的特点是在体外的自我剪接反应，作为一种手段，定位域5结合位点。结构域2、3、4或6的单一缺失不阻断任一剪接点处的体外反应，尽管结构域6的缺失在某种程度上降低了3'剪接位点选择的保真度。甚至缺少结构域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的缺失阻断3'剪接点处的反应，但不阻断5'剪接点处的反应。根据这些结果，我们得出结论，结构域5的结合位点在结构域1内，并且5'外显子、结构域1和结构域5（加上短连接序列）的复合物构成该内含子的基本催化核心。
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. A trans assay 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 5-gamma of the coxI gene 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.