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，涉及的分子机制包括 RNA聚合酶II对启动子的识别和利用还不彻底 明白了。我们建议在这里扩展我们最近的几项研究 RNA聚合酶的起始和向伸长的转变II.我们 已经表明，流产的启动伴随着生产性的启动 聚合酶。流产启动与生产启动的比率(即， 启动子清除效率)在很大范围内 各种各样的推动者。在某些情况下，但不是所有情况下，较弱的推动者 以不那么有效的清除为特点。我们现在建议解决 以下是问题。刺激性转录因子的使用 提高效率低下的推广者的通关效率？ 一些核心转录因子的高水平是否会抑制 失败的入会？我们能不能理解 从启动子序列和启动子序列看启动子的流产起始 建筑？特别是，塔塔盒子和塔塔的相对作用是什么 决定启动子清除效率的启动子元件？ 人们曾假设，一旦实现启动子清除，RNA 聚合酶II转录复合体具有相对统一的结构 在整个延伸过程中。我们最近表明，情况并非如此， 无论是在结构上还是在稳定性方面，对于高度纯化的络合物 在拉伸过程中在两个不同的位置暂停。我们将集结并 表征其他暂停的延长复合体，包括暂停的复合体 在自然停顿地点，以确定结构特征是否 可以与高概率的暂停或低复杂性相关 稳定性。我们还展示了一个应该被识别的序列 作为延伸过程中的停顿位置，在下列情况下不会导致明显的停顿 非常接近转录起始点的。我们会 构建一系列启动子，在其中放置潜在的停顿位置 在离起爆地点越来越远的地方，以便绘制 暂停位置生效所需的最小伸长程度。 最后，我们将尝试绘制RNA聚合酶自身的区域图 它们对引发、清除和有效伸长都很重要。 这些合作研究将涉及描述行为的特征， 特别是在启动和清除现有的温度敏感型 酵母RNA聚合酶及其新菌株的产生和性质 酵母RNA聚合酶II中的突变。

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.