5 AND 3 END PROCESSING OF YEAST MITOCHONDRIAL PRE-MRNAS

线粒体的生物发生需要细胞核的协调表达， 和线粒体基因组。 长期目标是了解 酿酒酵母中的控制信号， 产生线粒体成分编码的单独的 隔间 核呼吸缺陷的特征 一种酵母菌，pet突变体，已经鉴定出几个核基因 线粒体转录物的适当加工所必需的， mRNA的翻译。 本提案的目的是调查 两种核编码的线粒体RNA加工酶在 调节细胞编码蛋白质的合成。 核 基因产物，最初通过PET突变鉴定， 负责在线粒体mRNA上赋予稳定的5'末端 对于细胞色素B，辅酶Q的唯一的经细胞编码的亚基， 细胞色素c还原酶 这种CBP 1（细胞色素B加工）蛋白将 通过柱色谱法进一步纯化，其作用将是 在体外研究。 在SP 6噬菌体中产生的RNA 启动子-转录系统将被用作底物。 的序列和 RNA底物被蛋白质识别的结构要求 将通过表征线粒体抑制因子来研究， 减轻cbp 1突变，并通过测试改变的RNA底物， 体外测定 另一种蛋白质将被分离出来， 将线粒体多基因初级转录物切割成单基因 节，从而赋予成熟的3'末端上的几个线粒体 mRNA。 该内切核酸酶的纯化将通过以下方式之一完成 两种方法。 蛋白质将通过常规方法纯化。 色谱法，采用体外功能测定， 人工SP 6产生的RNA底物。 使用针对这种抗体的抗体 蛋白质，编码该酶的核基因将选自cDNA 表达式库。 或者，将使用间接方法， 包括表征这种活性缺陷的PET突变体， 通过用克隆的野生型酵母DNA转化分离基因， 产生针对过表达的E. colitrpE-3 '加工 基因融合产物，并在整个免疫系统中对蛋白质进行免疫检测。 色谱程序。 为了确定编码这些的基因是否 蛋白质处于一个更高级的调节系统中， 几个细胞核编码的线粒体成分同时， 在特定的5 '端和一般的3'端产生的mRNA水平中， 当野生型酵母从 葡萄糖抑制条件对在不可发酵碳源上生长的影响。

Mitochondrial biogenesis requires the coordinate expression of the nuclear and mitochondrial genomes. The long term goal is to understand the nature of the controlling signals in Saccharomyces cerevisiae that synchronize the production of mitochondrial constituents encoded in the separate compartments. The characterization of nuclear respiratory deficient strains of yeast, pet mutants, has identified several nuclear genes necessary for proper processing of mitochondrial transcripts and translation of the mRNAs. The aim of this proposal is to investigate the role of two nuclearly encoded mitochondrial RNA processing enzymes in regulating the synthesis of mitochondrially encoded proteins. A nuclear gene product, initially identified by pet mutations, is specifically responsible for conferring a stable 5' terminus on the mitochondrial mRNA for cytochrome b, the only mitochondrially encoded subunit of coenzyme Q - cytochrome c reductase. This CBP1, (cytochrome b processing), protein will be further purified by column chromatography, and its action will be studied in vitro. RNAs produced in an SP6 bacteriophage promoter-transcription system will be used as substrates. The sequence and structural requirements of the RNA substrate for recognition by the protein will be investigated by characterizing mitochondrial suppressors that alleviate cbp1 mutations, and by testing altered RNA substrates in the in vitro assay. Another protein will be isolated that is responsible for cleaving mitochondrial multigenic primary transcripts into unigene segments, thereby conferring mature 3' termini on several mitochondrial mRNAs. Purification of this endonuclease will be accomplished by one of two approaches. The protein will be purified by conventional chromatographic methods, employing an in vitro functional assay with artificially SP6-generated RNA substrates. Using antibodies raised to this protein, the nuclear gene encoding the enzyme will be selected from a cDNA expression library. Alternatively, an indirect approach will be used, involving characterization of pet mutants defective in this activity, isolation of the gene by transformation with cloned wild-type yeast DNA, production of an antibody to an overexpressed E. coli trp E-3'processing gene fusion product, and immuno-detection of the protein throughout a chromatographic procedure. To ascertain whether the genes encoding these proteins are under a higher order regulatory system that coordinates several nuclearly encoded mitochondrial components simultaneously, changes in the levels of mRNAs produced for the specific 5'-end and general 3'-end endonucleases will be monitored when wild-type yeast is switched from glucose repression conditions to growth on a non-fermentable carbon source.