CONTROL OF TRANSCRIPTION OF ISOLATED EUKARYOTIC GENES

拟议实验计划的主要目标是阐明 复杂顺式作用元件介导激活的机制 酵母烯醇化酶基因ENO 2的表达。 GCR 1基因编码一种 高水平表达所需的正调控蛋白 包括ENO 2的酵母糖酵解基因的转录。 的序列 介导的GCR 1依赖性激活ENO 2表达已经被 鉴定 此调节元件（GCR 1依赖性UAS元件） 含有两个RAP 1蛋白结合位点和一个ABF 1结合位点 在调节转录中起重要作用的蛋白质 调节元件的活性。 GCR 1蛋白将被纯化， GCR 1蛋白的结合位点将在GCR 1依赖的 无人机系统。 GCR 1结合单独和与 ABF 1和RAP 1在调节截短的、突变的和完整的 将在体内测试GCR 1依赖性UAS元件的长度版本。 SGC 1基因的显性突变先前被鉴定为 由gcr 1引起的生长和转录缺陷的抑制因子 无效突变 SGC 1编码一个基本螺旋环螺旋的成员 （bHLH）DNA结合蛋白家族。 SGC 1 bHLH蛋白将 SGC 1 bHLH蛋白的纯化和结合位点将在 或邻近GCR 1依赖的UAS元件。 SGC 1 bHLH的作用 蛋白结合，单独和与ABF 1，RAP 1和GCR 1组合， 将研究调节GCR 1依赖性UAS元件的活性 以确定这些蛋白质如何联合调节 ENO 2的表达。 GCR 1、SGC 1和 RAP 1调节ENO 2 UAS元件依赖的转录激活将 使用体外转录测定和纯化的GCR 1进行进一步研究， SGC 1和RAP 1蛋白。 两种分子的转录活性 由两种不同的GCR 1转录物编码的GCR 1蛋白的形式将是 比较了 将筛选gcr 1抑制突变体的集合， 区分新的基因，调节转录活性的 来自ENO 2的GCR 1依赖性UAS元件。 最后，最近的实验表明， 酵母SIN 3和SIN 4基因产物共同发挥作用， REB 1和RAP 1 DNA蛋白的生化特性。 遗传 研究表明，sin 3或sin 4无效突变显著改变了 RAP 1依赖性UAS元件的生物活性，REB 1依赖性UAS元件的生物活性， ENO 1 URS元件和REB 1依赖性酵母核糖体 增强子/终止子元件。 虽然我们对他们的情况知之甚少， 作用机制，REB 1和RAP 1似乎是全球调节剂， 酵母基因表达（RAP 1调节GCR 1依赖的 UAS元件）。 提出了实验研究 SIN 3和SIN 4蛋白调节蛋白质活性的机制 REB 1和RAP 1。 这项调查的长期目标是了解 多种调节蛋白协同作用的机制， 调节真核基因表达。

The primary objective of the proposed experimental plan is to elucidate the mechanism by which a complex cis-acting element mediates activation of expression of the yeast enolase gene ENO2. The GCR1 gene encodes a positive regulatory protein which is required for high level transcription of yeast glycolytic genes including ENO2. Sequences that mediate GCR1-dependent activation of ENO2 expression have been identified. This regulatory element (GCR1-dependent UAS element) contains two binding sites for RAP1 protein and a binding site for ABF1 protein that play important roles in modulating the transcriptional activity of the regulatory element. GCR1 protein will be purified and binding sites for GCR1 protein will be mapped within the GCR1-dependent UAS element. The role of GCR1 binding, alone and in combination with ABF1 and RAP1, in modulating the activity of truncated, mutant, and full length versions of the GCR1-dependent UAS element will be tested in vivo. Dominant mutations in the SGC1 gene were previously identified as suppressors of both growth and transcriptional defects caused by a gcr1 null mutation. SGC1, encodes a member of the basic-helix-loop-helix (bHLH) family of DNA binding proteins. SGC1 bHLH protein(s) will be purified and binding sites for SGC1 bHLH protein(s) will be mapped within or adjacent to the GCR1-dependent UAS element. The role of SGC1 bHLH protein binding, alone and in combination with ABF1, RAP1, and GCR1, in modulating the activity of the GCR1-dependent UAS element will be studied in vivo to determine how these proteins act in combination to regulate activation of ENO2 expression. The mechanism whereby GCR1, SGC1, and RAP1 modulate ENO2 UAS element-dependent activation of transcription will be further studied using in vitro transcription assays and purified GCR1, SGC1, and RAP1 proteins. The transcriptional activities of two molecular forms of GCR1 protein encoded by two distinct GCR1 transcripts will be compared. A collection of gcr1 suppressor mutants will be screened to distinguish novel genes that modulate the transcriptional activity of the GCR1-dependent UAS element from ENO2. Finally, recent experiments show that the yeast SIN3 and SIN4 gene products function together to regulate the biochemical properties of REB1 and RAP1 DNA proteins. Genetic studies showed that sin3 or sin4 null mutations dramatically altered the biological activities of a RAP1-dependent UAS element, the REB1-dependent ENO1 URS element, and the REB1-dependent yeast ribosomal enhancer/terminator element. Although little is known about their mechanism of action, REB1 and RAP1 appear to be global regulators of yeast gene expression (RAP1 modulates the activity of the GCR1-dependent UAS element described above). Experiments are proposed to investigate the mechanism whereby SIN3 and SIN4 proteins regulate the activities of REB1 and RAP1. The long term goal of this investigation is to understand the mechanisms whereby multiple regulatory proteins act in concert to modulate eucaryotic gene expression.