Mechanism of Group II Intron Retrotransposition

描述(申请人提供)：可移动的第二类内含子是被广泛假设与非LTR反转录转座子和剪接体内含子相关的逆转录元件。这个项目的总体目标是结合遗传、生化和结构研究来表征乳酸乳球菌第二组内含子编码蛋白(LTRA)和参与内含子移动的途径，特别是逆转录转座。要实现这一点，有两个具体目标。首先，将对LTRA的结构域进行详细的生化分析。我们将探索成熟酶、内切酶和逆转录酶活性与结构域的关系，以了解LTRA的模块组成和进化。孤立的结构域也将被证明对结晶学有用。其次，为了更好地了解内含子逆转录转座的机制，我们将使用一种可选择的遗传分析方法来检测第II组乳酸乳杆菌内含子LtrB的单个逆转录转座事件。不同的质粒受体将被用来探索内含子转座的核酸靶标以及对复制和/或转录过程的依赖性。因此，我将了解第二组内含子生物学的基本原理，以及它们与非LTR反转录转座子和剪接体内含子的适当关系。

DESCRIPTION (provided by applicant): Mobile group II introns are retroelements that are widely hypothesized to be related to both non-LTR retrotransposons and spliceosomal introns. The overall goal of this project is to combine genetic, biochemical and structural studies to characterize the Lactococcus lactis group II intron-encoded protein (LtrA) and the pathways involved in intron mobility, particularly retrotransposition. To accomplish this, there are two specific aims. First, detailed biochemical analyses on the structural domains of LtrA will be performed. The relationship of the maturase, endonuclease, and reverse transcriptase activities to domains will be explored to understand the modular composition and evolution of LtrA. Isolated domains will also prove useful for crystallography. Second, to better understand the mechanisms of intron retrotransposition, a selectable genetic assay for isolated retrotransposition events of the group II L. lactis intron LtrB will be used. Different plasmid recipients will be employed to explore the nucleic acid targets of intron transposition and the dependency on the processes of replication and/or transcription. I will therefore understand the basic principles of group II intron biology and their relationship, as appropriate, to non-LTR retrotransposons and spliceosomal introns.