GENETIC AND MOLECULAR STUDIES OF GROUP II INTRONS

描述：本续期申请的重点是结构和 aI 2的功能，一个来自酵母线粒体的自我剪接内含子。在 以前的工作表明，ORF 1）是一个成熟酶，2）是一个位点， 特异性移动的元件，和3）含有与逆转录酶的同源性 转录酶（RT）、DNA内切核酸酶（Zn）和逆转录病毒蛋白酶 （P）。有三个主要的具体目标。1)aI 2内含子表达为 作为96 kD前体，并加工成含有RT、X和Zn的p62。 申请人将确定p62是否也包含P和Z， 在一些非LTR逆转录元件中，以及这些序列是否重要 用于拼接和/或移动性。2)确定成熟酶的作用 在拼接中，提出了两种方法。 首先，PI将测试 p62促进内含子正确折叠的特定假设， 寻找RNA-蛋白质相互作用，使用各种标准 技术.与其他蛋白质的结合也将使用 p62作为几种RNP沉积的知识。这些生化 将利用从《公约》获得的资料对这些办法进行补充， 创造出一种新的突变体和抑制子。其次，他将试图 开发一个p62依赖的剪接系统，基于这样一个事实， 在体外低盐条件下不自剪接。正向和反向剪接 描述了测定。3)第二组内含子迁移的工作模型 将进行测试，其中涉及aI 2- 含有前mRNA和位点特异性插入受体， 无内含子基因除了RT之外，Zn也是必不可少的，可能通过 为第一链cDNA合成提供引物。稳获 RNA中间体的情况下将明确测试一个la Boeke， 芬克剪接与迁移率的关系将通过以下方法进行研究： 突变体的分析，包括切除的 intron参与其中。外显子序列在受体和供体中的作用 将被区分，体内移动性要求将被 与新的体外核酸内切酶测定（待进行）相比， Lambowitz实验室）。最后，申请人将调查 不寻常的移动性事件，可能涉及RT非依赖性途径， 反向剪接依赖途径。

DESCRIPTION: The focus of this renewal application is the structure and function of aI2, a self-splicing intron from yeast mitochondria. In previous work it was shown that the ORF 1) is a maturase, 2) is a site- specific mobile element, and 3) contains homology to reverse transcriptases (RT), DNA endonucleases (Zn), and retroviral proteases (P). There are three major specific aims. 1) The aI2 intron is expressed as a 96kD precursor, and processed to p62 which contains RT, X and Zn. The applicant will determine if p62 also contains P and Z, a domain found in some non-LTR retroelements, and whether these sequences are important for splicing and/or mobility. 2) To determine the role of the maturase in splicing, two approaches are proposed. First, the PI will test the specific hypothesis that p62 promotes proper folding of the intron by looking for RNA-protein interactions using a variety of standard techniques. Associations with other proteins will also be assayed using the knowledge that p62 sediments as several RNPs. These biochemical approaches will be supplemented with information obtained from the creation of aI mutants and suppressors. Second, he will attempt to develop a p62-dependent splicing system, based on the fact that aI does not self-splice at low salt in vitro. Both forward and reverse splicing assays are described. 3) A working model for group II intron mobility will be tested which involves reverse transcription of the aI2- containing pre-mRNA and site-specific insertion into recipient, intronless genes. In addition to RT, Zn is essential, perhaps by providing the primer for first-strand cDNA synthesis. The presumptive case for an RNA intermediate will be definitively tested a la Boeke and Fink. The relationship of splicing to mobility will be investigated by the analysis of mutants, including the possibility that the excised intron is involved. The role of exon sequences in recipient and donor will be distinguished and the in vivo mobility requirements will be compared to those for a new in vitro endonuclease assay (to be carried by the Lambowitz laboratory). Finally, the applicant will investigate unusual mobility events which may involve an RT-independent pathway and a reverse-splicing-dependent pathway.