Single molecule studies of nucleosomes and transcription

真核细胞染色质中的DMA是高度堆积的，但DNA中包含的信息必须以一种受调节的方式被细胞机器所获取，细胞机器对其进行解码，以制造作为细胞主要催化和结构成分的蛋白质。解码过程的第一步，转录，是由RNA聚合酶完成的。RNA聚合酶作为一个高度进程的分子马达，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接近被包装在核小体中的DNA，而转录在一定程度上是由DNA的可接近性来调节的。转录的

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.