Physical Chemistry of Nucleic Acids

描述(申请人提供)：转录是细胞中基因表达的中心控制点。真核细胞中的POL II酶必须在核小体DNA存在的物理屏障存在的情况下转录，并且该酶和它的原核对应物都必须转录受扭转限制的DNA片段，这些DNA片段需要酶针对增加的扭矩进行移位。此外，在转录延伸过程中，这两种结构高度同源的酶执行一个机械力化学循环，在这个循环中，核苷酸缩合反应与运动、力和扭矩产生的机械任务相耦合。在这一应用中，我们建议使用成像和单分子操作相结合的方法来：a)研究酵母Pol II通过核小体DNA进行转录的动力学。b)确定延伸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动力学的影响。我们还建议继续我们之前对原核生物大肠杆菌RNA聚合酶的研究，以研究：a)它在扭转限制的DNA转录过程中产生的扭矩，以及b)它在高空间分辨率下的机械力化学循环的细节。与公共健康相关：转录是基因表达调控的中心控制点。这一过程的中断与先天性心脏病、肿瘤发生和多种发育病理有关。我们在转录方面的实验提供的见解将支持未来疗法开发的第一步。

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.