Physical Chemistry of Nucleic Acids

描述（由申请人提供）：转录构成细胞中基因表达控制的中心点。真核Pol II酶必须在核小体DNA中存在物理屏障的情况下进行转录，并且这种酶和它的原核对应物都必须转录扭转受限的DNA片段，这需要酶在增加扭矩的情况下进行易位。此外，在转录延伸期间，这两种结构高度同源的酶执行机械化学循环，其中核苷酸缩合反应与运动，力和扭矩产生的机械任务相耦合。在这个应用中，我们建议使用成像和单分子操作方法的组合来：a)研究酵母Pol II通过核小体DNA的转录动力学。b)确定伸长因子对核小体转录的影响。c)研究组蛋白修饰对核小体转录动力学的影响。d)通过核小体DNA研究重塑因子对Pol II动态的影响。我们还建议继续我们先前对原核大肠杆菌RNA聚合酶的研究，以研究：a)其在扭转约束DNA转录过程中产生的扭矩，以及b)其机械化学循环的高空间分辨率细节。公共卫生相关性：转录构成基因表达调控控制的中心点。这一过程的破坏与先天性心脏病、肿瘤发生和多种发育病理学有关。我们的实验提供的关于转录的见解将支持未来治疗发展的第一步。

DESCRIPTION (provided by applicant): Transcription constitutes the central point of control for gene expression in the cell. The eukaryotic Pol II enzyme must transcribe in the presence of the physical barriers present in nucleosomal DNA and both this enzyme and its prokaryotic counterpart must transcribe torsionally constrained DNA segments that require the enzymes to translocate against increasing torques. Moreover, during transcription elongation these two highly structurally homologous enzymes perform a mechanochemical cycle in which a nucleotide condensation reaction is coupled to the mechanical tasks of movement, force, and torque generation. In this application we propose to use a combination of imaging and single molecule manipulation approaches to: a) Investigate the dynamics of transcription through nucleosomal DNA by yeast Pol II. b) Determine the effect of elongation factors on nucleosomal transcription. c) Investigate the effect of histone modification on the dynamics of nucleosomal transcription. d) Investigate the effect of remodeling factors on the dynamics of Pol II through nucleosomal DNA. We also propose to continue our previous studies of the prokaryotic E. coli RNA polmerase enzyme to investigate: a) Its generation of torque during transcription of torsionally constrained DNA, and b) The details of its mechanochemical cycle at high spatial resolution. PUBLIC HEALTH RELEVANCE: Transcription constitutes the central point of control for regulation of gene expression. Disruption of this process is associated with congenital heart disease, oncogenesis, and a large variety of developmental pathologies. The insights that our experiments provide on transcription will support the first steps in the development of future therapies.