TRNA PROCESSING NUCLEASES FROM LIVER MITOCHONDRIA

肝线粒体5‘和3’tRNA加工核酸酶 将使用目前的酶方法进一步纯化 净化。这些天然形式的分子大小 两种加工核酸酶将通过沉降法进行测定 分析和分子筛分技术，以及 蛋白质组分将用十二烷基硫酸钠-聚丙烯酰胺凝胶测定 电泳法。高纯度酶的功能研究 将使用正常和改变的线粒体(32P)进行 线粒体DNA片段体外合成的前tRNA转录本 克隆到转录载体中或从合成模板中克隆 含有T7 RNA聚合酶启动子。最优的 将确定底物的反应条件和光谱 使用正常的线粒体tRNA前体转录本，以及 前体底物的序列/结构特征是 本课程将考察识别和有效处理的关键 用化学方法产生的改变的前tRNA转录本 合成的模板。加工(32便士)产品将被 在聚丙烯酰胺/尿素凝胶上分离，提取并分析 使用一次和二次的常规指纹识别 摘要和端基分析。《3‘》的活动 加工核酸酶将用(32P)5‘-加工的tRNA进行检测 中间体。将确定线粒体3‘是否 加工核酸酶是一种精确的核酸内切酶，与 5‘端酶的活性。一种基本核糖核酸的存在 与5‘和3’处理核酸酶相关的成分将 通过这些酶对微球菌核酸酶的敏感性进行测试 消化。核糖核酸组分(S)将被分离和测序 通过对目前的RNA测序蛋白质组来确定可能的 核糖核酸的起始点(线粒体或核)(S)。在……里面 此外，胞质(线粒体外)5‘处理 核酸酶将进一步提纯，以便与 线粒体对应物。

The 5' and 3' tRNA processing nucleases from liver mitochondria will be further purified using current methods of enzyme purification. The molecular sizes of the native forms of these two processing nucleases will be measured by sedimentation analysis and moleuclar sieving techniques, and the multiplicity of protein components will be determined by SDS-polyacrylamide gel electrophoresis. Functional studies of the highly purified enzymes will be performed using normal and altered mitochondrial (32P) pre-tRNA transcripts produced in vitro from mtDNA fragments cloned into transcription vectors, or from synthetic templates containing the T7 RNA polymerase promoter. The optimal reaction conditions and spectrum of substrates will be determined using normal mitochondrial tRNA precursor transcripts, and the sequence/structural features of precursor substrates that are critical to recognition and efficient processing will be examined with altered pre-tRNA transcripts produced from chemically synthesized templates. Processed (32P) products will be separated on polyacrylamide/urea gels, excised, and analyzed by conventional fingerprinting using primary and secondary digestions, and end-group analyses. The activity of the 3' processing nuclease will be assayed with (32P) 5'- processed tRNA intermediates. It will be determined whether the mitochondrial 3' processing nuclease is a precise endonuclease, consistent with the activity of the 5' enzyme. The existence of an essential RNA component associated with the 5' and 3' processing nucleases will be tested by sensitivity of these enzymes to micrococcal nuclease digestion. The RNA component(s) will be isolated and sequenced by current RNA sequencing proteocols to determine the probable site of origin (mitochondrial or nuclear) of the RNA(s). In addition, the cytosolic (extra-mitochondrial) 5' processing nuclease will be further purified for direct comparison with the mitochondrial counterpart.