Physical Chemistry of Nucleic Acids

描述(申请人提供)：转录是生命的基本过程，构成细胞中基因表达的第一步，也是控制关键生物学事件的关键检查点，包括细胞生长、分化和肿瘤形成。在真核生物中， 这一高度调控的过程是由RNA聚合酶II(POL II)执行的。为了转录DNA，这种酶必须克服遗传物质核小体组织施加的物理障碍。此外，POL II必须转录受扭力限制的DNA片段，这些DNA片段需要酶在不断增加的扭矩下移位。多种机制参与调控转录，包括染色质的结构变化、各种转录因子、聚合酶产生扭矩的能力以及依赖于ATP的染色质重塑复合体的活性。在这项应用中，我们建议使用单分子操作和可视化方法相结合的方法来表征这些转录控制机制。具体地说，我们建议：1)通过核小体阵列研究转录的动力学，以阐明核小体间元件对屏障的贡献。2)确定转录因子IIS(TFIIS)和IIF(TFIIF)促进核小体转录的机制。3)描述DNA的扭转应变与POL II通过核小体转录的能力之间的关系。4)表征SWI/SNF复合体的核小体重塑机制、力化学性质和扭矩产生。

DESCRIPTION (provided by applicant): Transcription is an essential process for life that constitutes the first step of gene expression in the cell and a crucial checkpoint for the control f critical biological events, including cell growth, differentiation, and oncogenesis. In eukaryotes, this highly regulated process is carried out by RNA polymerase II (Pol II). To transcribe the DNA, this enzyme must overcome the physical barriers imposed by the nucleosomal organization of the genetic material. Moreover, Pol II must transcribe torsionally constrained DNA segments that require the enzyme to translocate against increasing torques. Various mechanisms participate in the control and regulation of transcription, including structural changes of chromatin, various transcription factors, the polymerase ability to generate torque, and the activity of ATP-dependent chromatin remodeler complexes. In this application we propose to characterize these mechanisms of transcription control using a combination of single molecule manipulation and visualization approaches. Specifically, we propose: 1) Investigate the dynamics of transcription through nucleosomal arrays to elucidate the contribution of internucleosomal elements to the barrier. 2) Determine the mechanisms by which transcription factors IIS (TFIIS) and IIF (TFIIF) enhance nucleosomal transcription. 3) Characterize the relationship between the torsional strain in DNA and the ability of Pol II to transcribe through a nucleosome. 4) Characterize the nucleosome-remodeling mechanism, mechanochemical properties, and torque generation of the SWI/SNF complex.