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中存在的物理屏障的存在下转录，并且这种酶及其原核对应物必须转录扭转约束的DNA片段，这需要酶针对增加的扭矩进行易位。此外，在转录延伸过程中，这两种高度结构同源的酶进行机械化学循环，其中核苷酸缩合反应与运动，力和扭矩产生的机械任务相结合。在本申请中，我们提出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以前对原核E.大肠杆菌RNA聚合酶研究：a）在扭转约束的DNA转录过程中产生的扭矩，和B）在高空间分辨率下的机械化学循环的细节。公共卫生相关性：转录是基因表达调控的中心点。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学有关。我们的实验提供的关于转录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.