COMPLEX LOCUS INVOLVED IN CYTOCHROME B MRNA MATURATION

线粒体的生物发生需要协调表达 核基因组和线粒体基因组。这项研究的长期目标是 了解酵母菌控制信号的性质 与线粒体蛋白生产同步的酿酒酵母 编码在单独的隔间中。原子核的特性 呼吸缺陷宠物突变体已经确定了几个必要的基因 用于处理线粒体前体转录本和翻译 线粒体核糖体上的mRNAs。一组宠物突变体 描述了编码76,5000道尔顿蛋白的核基因CBP1 它被输入细胞器，并与细胞器5‘端相互作用 细胞色素b前体RNA。CBP1基因表现出复杂的模式 与碳有关的大量受调控的PolyA+RNA 消息来源。这项建议的具体目的是研究相互作用 Cbp1蛋白与细胞色素b前体RNA的体外结合 描述CBP1在体内的作用部位，并研究其在体内的作用 CBP1转录的调控。CBP1蛋白将被提纯并 它与T3产生的底物RNA的相互作用将在 体外培养。该蛋白质将从商业或实验室酵母中获得。 通过尺寸排除、离子交换和亲和力的组合 层析，或通过融合到酵母信息素的阿尔法因子信号 序列，或通过体外翻译T3产生的mRNA。活动 将通过各种RNA结合和断裂来监测蛋白质的变化 化验。GBP1在体内的作用部位将通过 细胞色素b 5‘端区的接头-扫描器突变。 吉恩。CBP1基因产生两种不同的PolA+RNAs：全长2.3 Kb的mRNA和1.3 kb的RNA。从基因的5‘半中分离出来。在切换时 酵母从发酵到呼吸生长条件下，2.3kb的RNA 减少伴随着1.3kb RNA的增加。如何以及为什么 RNAs的丰度受碳源的调节 通过突变研究进行研究。CBP1紧挨着市建局 (功能未知)和NUC1(线粒体核酸酶)基因。中的删除 URF上调CBP1的表达。URF和NUC1的影响 将通过缺失和插入的方式来研究CBP1的表达 诱变。RNA水平和5‘/3末端的变化将由 Northern分析和S1分析。

Mitochondrial biogenesis requires the coordinate expression of the nuclear and mitochondrial genomes. The long term goal of this research is to understand the nature of the controlling signals in Saccharomyces cerevisiae that synchronize the production of mitochondrial proteins encoded in the separate compartments. The characterization of nuclear respiratory deficient pet mutants has identified several genes necessary for processing of mitochondrial precursor transcripts and translation of the resultant mRNAs on mitochondrial ribosomes. One group of pet mutants describes the nuclear gene CBP1, which encodes a 76,5000 dalton protein that is imported into the organelle and interacts with the 5' end of the cytochrome b precursor RNA. The CBP1 gene exhibits a complex pattern of polyA+ RNAs which are regulated in abundance with respect to carbon source. The specific aims of this proposal are to study the interaction of CBP1 protein with precursor RNA for cytochrome b in vitro, to delineate the site of action of CBP1 in vivo, and to investigate the regulation of CBP1 transcription. The CBP1 protein will be purified and its interaction with T3 generated substrate RNAs will be studied in vitro. The protein will be obtained from commercial or laboratory yeast by a combination of size exclusion, ion exchange and affinity chromatography, or by fusion to the yeast pheromone alpha-factor signal sequence, or by in vitro translation of T3-generated mRNA. The activity of the protein will be monitored by a variety of RNA binding and scission assays. The site of action of GBP1 in vivo will be investigated via linker-scanner mutagenesis of the 5' end region of the cytochrome b. gene. The CBP1 gene produces two different polyA+ RNAs: a full length 2.3 kb mRNA and a 1.3 kb RNA. from the 5' half of the gene. On switching yeast from fermentative to respiratory growth conditions, the 2.3 kb RNA decreases with a concomitant increase in the 1.3 kb RNA. How and why the abundance of the RNAs is regulated with respect to carbon source will be investigated by mutational studies. CBP1 is closely flanked by the URF (unknown function) and NUC1 (mitochondrial nuclease) genes. A deletion in URF up-regulates CBP1 expression. The influence of URF and NUC1 expression on that of CBP1 will be studied via deletion and insertional mutagenesis. Changes in RNA levels and 5'/3 ends will be monitored by Northern and S1 analyses respectively.