CONTROL OF TRANSCRIPTION IN EUKARYOTIC CELLS

而转录起始是最重要的单一控制点 在真核基因表达中， RNA聚合酶II对启动子的识别和使用还不彻底 明白 我们建议在这里扩展我们最近的几项研究， 通过RNA聚合酶II起始和过渡到延伸。 我们 已经表明，流产的启动伴随着生产性启动， 聚合酶 流产与生产性启动的比率（即， 启动子清除的效率）在很宽的范围内变化， 各种促销员。 在一些但不是所有情况下，较弱的启动子是 以较低的有效清除为特征。 我们现建议处理 以下问题。 使用刺激转录因子 增加在其他无效启动子处的清除效率？ 高水平的核心转录因子是否会抑制 流产的启蒙？ 我们能否理解 启动子之间的失败启动，就其序列和 建筑？ 特别是，TATA盒的相对作用是什么， 启动子元件在决定启动子清除效率？ 曾经假设，一旦实现启动子清除， 聚合酶II转录复合物具有相对均匀的结构 在整个伸长过程中。 我们最近的研究表明情况并非如此， 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。 我们将集合起来， 表征其他暂停的延长复合物，包括暂停的复合物 在自然停顿点，为了确定结构特征是否 可以与高概率的暂停或低复杂度相关 稳定 我们还表明，一个应该被识别的序列 因为在伸长过程中的暂停部位不会引起明显的暂停， 非常靠近转录起始点。 我们将 构建一系列启动子，其中放置潜在的暂停位点 在距离起始位点增加的距离处，以绘制 暂停部位有效所需的最小伸长程度。 最后，我们将尝试绘制RNA聚合酶本身的区域 这对于引发、清除和有效伸长是重要的。 这些合作研究将包括描述行为， 特别是在启动和清除现有的温度敏感 酵母RNA聚合酶以及新的 酵母RNA聚合酶II内的突变。

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.