Mechanisms of eukaryotic transcription activation

转录激活是许多信号传递和发育途径的最终终点， 而理解激活机制是理解这些途径作用的关键。 从以前的研究来看，很明显，基因特异性突变对正常基因调控的破坏， 转录激活因子可导致癌症和其他疾病。这一广泛的长期目标 建议是确定基因特异性转录因子激活的机制， RNA聚合酶II（Pol II）。这项工作将为进一步了解基因组学提供基础 在分子水平上调节正常和疾病状态。 这项工作的具体目标将利用生物化学，结构和分子遗传学方法来检查 几种转录激活因子的直接靶点以及与这些靶点接触的机制 刺激转录。使用新开发的方法绘制蛋白质-蛋白质接触， 复合物，我们将确定直接目标的激活结构域在几个模型激活剂的存在或 没有染色质。将进行生物化学和分子遗传学研究，以测试 这些相互作用的转录。我们将扩展这些研究，以检查不同激活剂的靶点 类在TATA少启动子代理。在通过羟基自由基映射这些激活剂-靶标相互作用之后， 切割和直接蛋白质-蛋白质相互作用测定，我们将确定激活的结构 与其相关目标相结合。最后，通过阻止预启动的组装， 复杂的中间阶段，我们将研究的机制，激活剂接触之一， 这些目标，SAGA辅激活因子复合物，刺激转录。我们提出的工作将阐明 转录调控的重要机制和原则。

Activation of transcription is the ultimate endpoint for many signal transaction and developmental pathways, and understanding the mechanism of activation is a key to understanding the action of these pathways. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription activators can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms used by gene-specific transcription factors to activate transcription by RNA Polymerase II (Pol II). The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work will utilize biochemical, structural, and molecular genetic methods to examine the direct targets of several transcription activators and the mechanism whereby contact with these targets stimulates transcription. Using a newly developed method for mapping protein-protein contacts within large complexes, we will identify direct targets of activation domains in several model activators in the presence or absence of chromatin. Biochemical and molecular genetic studies will be conducted to test the relevance of these interactions in transcription. We will extend these studies to examine the targets of a different activator class acting at TATA-less promoters. After mapping these activator-target interactions by hydroxyl radical cleavage and direct protein-protein interaction assays, we will determine the structure of the activation domains in combination with their relevant targets. Finally, by blocking assembly of the Preinitiation Complex at intermediate stages, we will examine the mechanism by which activator contact with one of these targets, the SAGA coactivator complex, stimulates transcription. Our proposed work will illuminate important mechanisms and principles of transcriptional regulation.