5 AND 3 END PROCESSING OF YEAST MITOCHONDRIAL PRE-MRNAS

酵母线粒体前 MRNAS 的 5 和 3 末端处理

• 批准号: 3286701
• 负责人: Carol Louise Dieckmann
• 金额: $ 8.85万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286701
• 关键词: Saccharomyces; chromatography; cytochrome b; endonuclease; fungal genetics; gene expression; genetic library; genetic manipulation; genetic mapping; genetic recombination; genetic transcription; genetic translation; messenger RNA; mitochondria; molecular cloning; monoclonal antibody; mutant; nucleic acid sequence; radiotracer; structural genes

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.

线粒体的生物发生需要核的协调表达 和线粒体基因组。我们的长期目标是了解大自然 在酿酒酵母中控制信号的同步 分离编码的线粒体成分的产生 车厢。核呼吸虚证的特征 酵母菌株，宠物突变体，已经确定了几个核基因 对于线粒体转录本的正确处理和 MRNAs的翻译。这项提案的目的是调查 两种核编码的线粒体RNA加工酶在细胞周期中的作用 调节线粒体编码蛋白质的合成。一颗核子 基因产物，最初由pET突变鉴定，是特定的 负责赋予线粒体mRNA一个稳定的5‘末端 对于细胞色素b，辅酶Q的唯一线粒体编码亚基- 细胞色素c还原酶。这种CBP1，(细胞色素b处理)，蛋白质将 通过柱层析进一步纯化，其作用将是 在体外研究。SP6噬菌体产生的RNA 将使用启动子-转录系统作为底物。该序列和 蛋白质识别的RNA底物的结构要求 将通过表征线粒体抑制因子来进行研究 减轻cbp1突变，并通过检测in中改变的RNA底物 体外试验。另一种蛋白质将被分离出来，它负责 将线粒体多基因初级转录本切割成Unigene 片段，从而在几个线粒体上授予成熟的3‘末端 MRNAs。这种核酸内切酶的纯化将由下列方法之一完成 有两种方法。蛋白质将通过常规方法提纯 层析方法，采用体外功能测定 人工SP6产生的RNA底物。使用对此产生的抗体 蛋白质，编码该酶的核基因将从cDNA中选择 表达式库。或者，将使用间接方法， 涉及在这一活动中有缺陷的宠物突变体的特征， 用克隆的野生型酵母DNA转化分离该基因， 一株过表达的大肠杆菌Trp E-3‘加工抗体的制备 基因融合产品，以及整个过程中蛋白质的免疫检测 层析程序。为了确定编码这些基因的基因 蛋白质处于一个更高级别的调节系统中，它协调 几个核编码的线粒体成分同时发生变化 在为特定的5‘端和一般的3’端产生的mRNAs水平中 当野生型酵母从 在非发酵碳源上生长的葡萄糖抑制条件。