Single molecule studies of nucleosomes and transcription

描述(申请人提供)真核细胞染色质中的DNA是高度密集的，但DNA中包含的信息必须以一种受监管的方式被细胞机制获取，细胞机器对其进行解码，以产生作为细胞的主要催化和结构成分的蛋白质。这个解码过程的第一步，转录，是由RNA聚合酶执行的，它的功能是一个高度进行性的分子马达，沿着DNA模板移动，将遗传信息从DNA转录到信使RNA。在转录过程中，RNA聚合酶和其他转录因子必须能够访问包装在核小体中的DNA，而转录在一定程度上受到DNA的可及性的调节。转录在 染色质及其调控必须是涉及定向分子运动的高度动力学过程。因此，我们研究的长期目标是在动力学和机械水平上了解(1)包装在核小体中的DNA被RNA聚合酶和其他转录因子获取的许多过程，(2)各种RNA聚合酶如何转录核小体DNA，以及(3)这些过程中涉及的调节因子的功能。在这个项目中，我们建议使用PI实验室最近开发的新颖而强大的单分子生物物理方法来直接测量其中一些分子事件。这项建议的具体目的集中在染色质重塑机器对核小体稳定性和DNA可及性的影响，RNA聚合酶如何克服核小体障碍，以及转录对核小体的影响。我们期待我们的研究对理解转录及其在染色质中的调控做出重大贡献。

DESCRIPTION (provided by applicant) The DMA in the chromatin of a eukaryotic cell is highly packed and yet the information contained in the DNA must be made accessible, in a regulated way, to the cellular machinery that decodes it to make the proteins that are the main catalytic and structural components of the cell. The first step in this decoding process, transcription, is carried out by RNA polymerase which functions as a highly processive molecular motor that moves along the DNA template transcribing genetic information from DNA to messenger RNA. During transcription, RNA polymerase and other transcription factors must be able to access the DNA that is packed into nucleosomes and transcription is in part regulated by the accessibility of the DNA. Transcription in chromatin and its regulation must necessarily be highly kinetic processes involving directed molecular motions. Therefore, the long-term goals of our research are to understand on a kinetic and mechanical level (1) the many processes by which the DNA that is packed in nucleosomes is made accessible to RNA polymerase and other transcription factors, (2) how various RNA polymerases transcribe nucleosomal DNA, and (3) the functioning of the regulatory factors involved in these processes. In this project, we propose to use novel and powerful single molecule biophysical approaches that have been recently developed in the Pi's lab to make direct measurements of some of these molecular events. The specific aims of this proposal focus on the effects of chromatin remodeling machines on nucleosomal stability and DNA accessibility, how RNA polymerase overcomes the nucleosome obstacle, and the effects of transcription on nucleosomes. We expect our studies to make significant contributions to the understanding of transcription and its regulation in chromatin.