CONTROL OF TRANSCRIPTION OF ISOLATED EUKARYOTIC GENES

拟议的实验计划的主要目标是阐明 复杂的顺式作用元件调节激活的机制 酵母烯醇化酶基因ENO2的表达。GCR1基因编码一种 高水平所需的阳性调节蛋白 酵母糖酵解基因的转录，包括ENO2。这些序列 介导依赖于GCR1的ENO2表达的激活 已确认身份。此监管要素(依赖于GCR1的UAS要素) 包含RAP1蛋白的两个结合部位和ABF1的一个结合部位 在转录调控中起重要作用的蛋白质 调节要素的活动。GCR1蛋白将被提纯并 GCR1蛋白的结合位点将被定位在依赖于GCR1的 无人机小分队。GCR1结合的作用，单独或与 ABF1和RAP1在调节截短、突变和完全基因活性中的作用 依赖于GCR1的UAS元件的长度版本将在体内进行测试。 Sgc1基因的显性突变此前被鉴定为 由gcr1引起的生长和转录缺陷的抑制因子 零突变。SGC1编码基本-螺旋-环-螺旋的一个成员 (BHLH)DNA结合蛋白家族。SGC1bHLH蛋白(S) 纯化的SGC1bHLH蛋白(S)的结合位点将在 或与依赖于GCR1的UAS元件相邻。SGC1bHLH的作用 单独和与ABF1、RAP1和GCR1结合的蛋白质结合 将研究调节依赖GCR1的UAS元件的活性 在体内确定这些蛋白质如何结合在一起调节 激活ENO2的表达。GCR1、SGC1和GCR1 RAP1调节eNO2-UAS元件依赖的转录意愿激活 利用体外转录实验和纯化的GCR1进行进一步研究， SGC1和RAP1蛋白。两个分子的转录活性 由两个不同的GCR1转录本编码的GCR1蛋白的形式将是 比较一下。将对gcr1抑制突变体的集合进行筛选以 区分调节细胞转录活性的新基因 来自ENO2的依赖于GCR1的UAS元素。最后，最近的实验表明 酵母SIN3和SIN4基因产物共同发挥调节作用 Reb1和RAP1 DNA蛋白的生化性质。遗传 研究表明，sin3或sin4零突变显著改变了 依赖RAP1的UAS元件Reb1依赖的生物学活性 ENO1 URS元件与依赖Reb1的酵母核糖体 增强器/终结器元素。尽管人们对它们的情况知之甚少 作用机制，Reb1和RAP1似乎是全球调节因子 酵母基因表达(RAP1调节依赖于GCR1的活性 上述UAS元件)。建议进行实验以研究 SIN3和SIN4蛋白调节血管紧张素转换酶活性的机制 Reb1和RAP1。这项调查的长期目标是了解 多个调节蛋白协同作用的机制 调节真核基因的表达。

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.