GENETIC AND MOLECULAR STUDIES OF GROUP II INTRONS

描述：本次续签申请的重点是结构和 酵母线粒体自剪接内含子AI2的功能。在……里面 以前的工作表明，ORF 1)是一个成熟酶，2)是一个位点- 特定的移动元件，以及3)包含同源反转 转录酶(RT)、DNA内切酶(锌)和逆转录病毒蛋白酶 (P)。有三个主要的具体目标。1)表达了AI2内含子 作为96kD的前体，加工成含有RT、X和锌的p62。 申请者将确定P62是否也包含P和Z，这是一个发现的域 在一些非LTR逆转录元件中，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 在拼接方面，提出了两种方法。首先，PI将测试 P62通过促进内含子正确折叠的特定假说 使用各种标准寻找RNA-蛋白质相互作用 技巧。与其他蛋白质的关联性也将用 P62以几个RNP形式沉积的知识。这些生物化学 方法将得到从 人工智能突变体和抑制子的创建。第二，他将试图 开发一个依赖于p62的剪接系统，基于人工智能确实如此 在体外低盐条件下不能自我剪接。正向剪接和反向剪接 描述了分析方法。3)第二类内含子迁移率的工作模型 将进行涉及AI2反转录的检测- 含有前信使核糖核酸和位点特异性插入受体， 无内含子基因。除了RT，锌也是必需的，也许是通过 为合成第一链cdna提供了引物。推定 RNA中间体的情况将在La Boeke和 芬克。将通过以下方式研究剪接与移动性的关系 对突变体的分析，包括切除的 这与内含子有关。外显子序列在受体和供体中的作用 将被区别对待，活体活动要求将是 与一种新的体外核酸内切酶检测方法(即将进行)相比 由兰博维茨实验室提供)。最后，申请者将进行调查 异常的运动事件，可能涉及非RT依赖的途径和 依赖反向剪接的途径。

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.