CONTROL OF TRANSCRIPTION IN EUKARYCTIC CELLS

真核细胞转录的控制

• 批准号: 2175536
• 负责人: Donal Luse
• 金额: $ 22.79万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175536
• 关键词: DNA directed RNA polymerase; DNA footprinting; HeLa cells; binding proteins; eukaryote; gene expression; gene mutation; genetic promoter element; nucleic acid sequence; protein reconstitution; protein structure; temperature sensitive mutant; tissue /cell culture; transcription factor

While transcription initiation is the most important single control point in eukaryotic gene expression, the molecular mechanisms involved in promoter recognition and use by RNA polymerase II are not thoroughly understood. We propose here to extend several of our recent studies on initiation and the transition into elongation by RNA polymerase II. We have shown that abortive initiation accompanies productive initiation by the polymerase. The ratio of abortive to productive initiation (that is, the efficiency of promoter clearance) varies over a wide range among various promoters. In some but not all cases the weaker promoters are characterized by less effective clearance. We now propose to address the following questions. Will the use of stimulatory transcription factors increase the efficiency of clearance at otherwise inefficient promoters? Will high levels of some of the core transcription factors suppress abortive initiation? Can we understand the intrinsic differences in abortive initiation among promoters in terms of their sequence and their architecture? In particular, what is the relative role of the TATA box and the initiator element in determining the efficiency of promoter clearance? It had been assumed that once promoter clearance is achieved, the RNA polymerase II transcription complex has a relatively uniform structure throughout elongation. We have recently shown that this is not the case, either structurally or in terms of stability, for highly purified complexes paused at two different locations during elongation. We will assemble and characterize other paused elongation complexes, including complexes paused at natural pause sites, in order to determine whether structural features can be correlated with a high probability of pausing or low complex stability. We have also shown that a sequence which should be recognized as a pause site during elongation does not cause significant pausing when placed very close to the point of transcription initiation. We will construct a series of promoters in which potential pause sites are placed at increasing distances from the initiation site in order to map the minimum extent of elongation required for pause sites to become effective. Finally, we will attempt to map regions within the RNA polymerase itself which are important for initiation, clearance and effective elongation. These collaborative studies will involve characterizing the behavior, particularly at initiation and clearance, of existing temperature-sensitive yeast RNA polymerases as well as the generation and characterization of new mutations within yeast RNA polymerase II.

虽然转录起始是最重要的单一控制点 在真核基因表达中，涉及的分子机制 RNA聚合酶II对启动子的识别和使用不彻底 明白了。 我们在此建议扩展我们最近的几项研究 RNA 聚合酶 II 的起始和向延伸的转变。 我们 已经表明，流产的起始伴随着生产性的起始 聚合酶。 失败启动与生产启动的比率（即 启动子清除的效率）在很大范围内变化 各种促销员。 在某些情况下，但并非所有情况下，较弱的启动子是 其特点是有效间隙较小。 我们现在建议解决 以下问题。 是否会使用刺激性转录因子 提高原本效率低下的启动子的清除效率？ 一些核心转录因子的高水平会抑制 启动失败？ 我们能否理解其中的内在差异？ 启动子之间的启动失败在其序列和它们的方面 建筑学？ 特别是TATA盒子的相对作用是什么？ 决定启动子清除效率的起始元件？ 人们曾假设，一旦启动子被清除，RNA 聚合酶II转录复合物具有相对均匀的结构 整个伸长率。 我们最近表明情况并非如此， 对于高度纯化的复合物，无论是在结构上还是在稳定性方面 在伸长过程中在两个不同的位置暂停。 我们将集合并 表征其他暂停伸长复合物，包括暂停复合物 在自然停顿位点，以确定结构特征是否 可以与高暂停概率或低复杂性相关 稳定。 我们还表明了一个应该被识别的序列 作为伸长过程中的暂停位点，不会导致显着的暂停 放置在非常靠近转录起始点的位置。 我们将 构建一系列启动子，其中放置潜在的暂停位点 离起始位点越来越远，以便绘制 暂停位点生效所需的最小伸长程度。 最后，我们将尝试绘制 RNA 聚合酶本身内的区域图 这对于引发、清除和有效伸长很重要。 这些合作研究将涉及行为特征， 特别是在现有温度敏感的启动和清除时 酵母RNA聚合酶以及新RNA聚合酶的产生和表征 酵母 RNA 聚合酶 II 内的突变。