CONTROL OF TRANSCRIPTION OF ISOLATED EUCARYOTIC GENES

本研究计划的目标是阐明 参与调节糖酵解和核糖体 酿酒酵母中的RNA基因。 独特的顺式作用 介导正或负调节的序列， 两个烯醇化酶基因（ENO 1和ENO 2）和一个 甘油醛-3-磷酸脱氢酶基因（TDH 3） 已被定位在每个基因的5'侧翼区域内。 与这些顺式作用的蛋白质形成稳定复合物 已经使用DNA/蛋白质鉴定了调节序列 结合测定。 遗传和生物化学研究表明， GCR 1基因产物，其是高效转录所需的 大多数糖酵解基因，干扰的结合， 阻遏物样蛋白的上游激活位点 ENO 2的序列（UAS）。 GCR 1蛋白和阻遏物 蛋白质将被纯化并广泛表征， 就其在协调调节ENO 1，ENO 2， 和TDH 3表达。 突变菌株在 阻遏物基因将被分离和表征。 利用这些 突变株和纯化的调节蛋白，相关性 将在特异性DNA/蛋白质结合事件之间进行， 研究中的糖酵解基因在生长于 糖酵解或产气碳源。 一种蛋白质， 与ENO 1中的上游阻遏序列（URS）的连接也将是 提纯 URS结合蛋白对结合的影响 其它调节蛋白质对ENO 1 UAS区域或TATAAA 将对序列进行研究。 突变菌株， 将分离URS结合蛋白基因中的缺陷， 表征了 一种高灵敏度的RNA检测方法 依赖聚合酶II的选择性转录起始 将开发vitro。 一旦进行体外转录测定， 它将与上述方法一起使用。 结合研究和遗传分析，以进一步阐明 参与调节糖酵解基因表达的机制。 体内合成34 SrRNA的序列要求将 进一步定义为使用携带 人工35 SrRNA基因。 这些研究将针对 建立邻近序列的转录功能， 34 SrRNA起始位点的存在和不存在下， 间隔子启动子序列，其刺激35 SrRNA合成， 体内10倍以上。 RNA聚合酶I所需的因子- 来自间隔区启动子的依赖性选择性转录将 被纯化并用于重建准确的转录， 体外

The goals of this research plan are to elucidate the mechanisms involved in regulating transcription of glycolytic and ribosomal RNA genes in Saccharomyces cerevisiae. Distinct cis-acting sequences which mediate positive or negative regulation of transcription of the two enolase genes (ENO1 and ENO2) and one of the glyceraldehyde-3-phosphate dehydrogenase genes (TDH3) have been mapped within the 5' flanking regions of each gene. Proteins which form stable complexes with these cis-acting regulatory sequences have been identified using DNA/protein binding assays. Genetic and biochemical studies suggest that the GCR1 gene product, which is required for efficient transcription of most glycolytic genes, interferes with the binding of a repressor-like protein to a site within the upstream activation sequences (UAS) of ENO2. The GCR1 protein and the repressor protein will be purified and extensively characterized with respect to their roles in coordinate regulation of ENO1, ENO2, and TDH3 expression. Mutant strains which are defective in the repressor gene will be isolated and characterized. Utilizing these mutant strains and the purified regulatory proteins, correlations will be made between specific DNA/protein binding events and expression of the glycolytic gene under study in cells grown on glycolytic or gluconeogenic carbon soruces. A protein which binds to the upstream repression sequences (URS) in ENO1 will also be purified. The effects of the URS binding protein on the binding of other regulatory proteins to the ENO1 UAS regions or TATAAA sequences will be investigated. Mutant strains which are defective in the URS binding protein gene will be isolated and characterized. A highly sensitive assay for detecting RNA polymerase II-dependent selective initiation of transcription in vitro will be developed. Once an in vitro transcription assay is available, it will be used in conjunction with the aforementioned binding studies and genetic analyses to further elucidate the mechanisms involved in regulating glycolytic gene expression. The sequence requirements for synthesis of 34SrRNA in vivo will be further defined using a centromere plasmid carrying an artificial 35SrRNA gene. These studies will be aimed toward establishing the transcriptional function of sequences adjacent to the 34SrRNA initiation site in the presence and absence of a spacer promoter sequence which stimulates 35SrRNA synthesis in vivo more than 10-fold. Factors required for RNA polymerase I- dependent selective transcription from the spacer promoter will be purified and used to reconstitute accurate transcription in vitro.