TRNA PROCESSING NUCLEASES FROM LIVER MITOCHONDRIA

肝线粒体5'和3' tRNA加工核酸酶 将使用现有的酶法进一步纯化 洁净. 这些天然形式的分子大小 将通过沉降测量两种加工核酸酶 分析和分子筛技术，以及 采用SDS-聚丙烯酰胺凝胶电泳法测定蛋白质组分 电泳 高纯度酶的功能研究 将使用正常和改变的线粒体（32 P）进行 从mtDNA片段体外产生的前tRNA转录物 克隆到转录载体中，或从合成模板 含有T7 RNA聚合酶启动子。 最优 确定了反应条件和底物光谱 使用正常的线粒体tRNA前体转录本， 前体底物的序列/结构特征， 识别和有效处理的关键将被检查 通过化学方法产生的改变的前tRNA转录本 合成模板 已加工（32 P）产品将 在聚丙烯酰胺/尿素凝胶上分离，切下，并通过 使用初级和次级的常规指纹识别 digestion和端基分析。 3'的活性 将用（32 P）5 ′-加工tRNA测定加工核酸酶 中间体的 将确定线粒体3' 加工核酸酶是一种精确的内切核酸酶，与 5'酶的活性。 一种重要RNA的存在 与5'和3'加工核酸酶相关的组分将 通过这些酶对微球菌核酸酶的敏感性进行测试 消化. 将对RNA组分进行分离和测序 通过目前的RNA测序蛋白酶来确定可能的 RNA的起源位点（线粒体或核）。 在 此外，胞质（线粒体外）5'加工 核酸酶将进一步纯化，用于与 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.