CONTROL OF TRANSCRIPTION OF ISOLATED EUCARYOTIC GENES

这项研究计划的目标是阐明这种机制。 参与调节糖酵解和核糖体的转录 酿酒酵母中的RNA基因。截然不同的顺势作用 介导正或负调控的序列 两个烯醇酶基因(ENO1和ENO2)和一个基因的转录 甘油醛-3-磷酸脱氢酶基因(TDH3) 已被定位在每个基因的5‘侧翼区域内。 与这些顺式作用蛋白形成稳定的络合物的蛋白质 已使用DNA/蛋白质鉴定了调控序列 有约束力的分析。遗传和生化研究表明， GCR1基因产物，这是有效转录所必需的 在大多数糖酵解基因中，会干扰一种 抑制器样蛋白结合到上游激活中的一个位置 ENO2的序列(UA)。GCR1蛋白与阻遏因子 蛋白质将被提纯并广泛鉴定为 关于它们在ENO1、ENO2、 和TDH3的表达。有缺陷的突变菌株 阻遏基因将被分离和鉴定。利用这些 突变菌株与纯化的调控蛋白的相关性 将在特定的DNA/蛋白质结合事件和 糖酵解基因在体外培养细胞中的表达 糖酵解或生糖碳山梨糖。一种结合在一起的蛋白质 到ENO1中的上游抑制序列(UR)也将是 纯净的。尿路蛋白结合蛋白对结合蛋白的影响 ENO1 UAS区或TATAAA的其他调节蛋白 将对序列进行调查。突变菌株，它们是 URS结合蛋白基因缺陷将被分离并 特色化的。一种检测RNA的高灵敏度方法 依赖于聚合酶II的选择性转录启动 体外培养将会被开发出来。一旦体外转录测试 如果可用，它将与上述 结合研究和遗传分析以进一步阐明 调节糖酵解基因表达的机制。 体内合成34SrRNA的序列要求如下 使用携带有一种 人工35SrRNA基因。这些研究的目的将是 建立相邻序列的转录功能 34SrRNA在存在和不存在的情况下的起始点 刺激35SrRNA合成的间隔区启动子序列 活体增加了10倍以上。RNA聚合酶I所需的因子- 间隔区启动子的依赖选择性转录将 被提纯并用于重建准确的转录 体外培养。

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.