CONTROL OF TRANSCRIPTION IN EUKARYOTIC CELLS

这项研究的长期目标是了解 真核生物RNA的合成。 总体战略是 涉及在无细胞系统中的转录。 提出要 从两个方面扩展和改进现有的体外方法。 一是在 为了定义和表征大分子复合物， 调节事件的靶点，一系列转录复合物， 将制备DNA模板和RNA聚合酶II。 两人都是新上任的 和延伸复合物将被研究;在所有情况下，模板保护 并检测RNA聚合酶构型和亚基组成。 还将进行研究以确定RNA聚合酶II的亚群 仅参与伸长的亚基。 第二，为了更 忠实地再现体内转录水平和模式， 生理模板比目前使用的纯化的DNA将是 制备 在这部分的研究中，我们将继续我们目前的研究， 研究重组染色质模板;我们还将准备 染色质模板从细胞中接近其天然状态， 可能 目的基因与牛乳头状瘤之间的相互作用 病毒将作为附加体在小鼠细胞中制备和繁殖。 基因 这样的构建体继续被适当地调节， 微型染色体 这些纯化的附加体将用作模板， 体外RNA合成，目的是证明体外转录 水平与相应的体内水平成比例。 两 金属诱导和发育调节 （β-珠蛋白）启动子将用于构建体中。 对于这些案件， 其在体内转录模式可以被复制，微型染色体 将检查感应相关特征。 这些措施包括： (i)结合在启动子或其它位点的调节蛋白的存在， 已知的调节位点（增强子），和（ii）构型的变化 启动子（不存在核小体，存在变性区域， 等）。

The long-term goal of this research is to understand the regulation of eukaryotic RNA synthesis at the molecular level. The general strategy to be employed involves transcription in cell-free systems. It is proposed to extend and improve the existing in vitro approaches in two ways. First, in order to define and characterize the macromolecular complex which is the target of regulatory events, a series of transcription complexes containing DNA template and RNA polymerase II will be prepared. Both newly-initiated and elongating complexes will be studied; in all cases template protection and RNA polymerase configuration and subunit composition will be examined. Studies will also be done to determine the subset of RNA polymerase II subunits involved solely in elongation. Second, in order to more faithfully reproduce in vivo transcription levels and patterns, more physiological templates than the currently-used purified DNAs will be prepared. In this part of the research, we will continue our current studies on reconstituted chromatin templates; we will also prepare chromatin templates from the cell in as close to their native state as possible. Recombinants between genes of interest and bovine papilloma virus will be made and propagated as episomes in mouse cells. Genes in such constructs continue to be properly regulated and may be recovered as minichromosomes. These purified episomes will be used as templates for in vitro RNA synthesis with the intent of demonstrating in vitro transcription levels proportional to the corresponding in vivo levels. Both metal-inducible (metallothione) and developmentally regulaterd (Beta-globin) promoters will be used in the constructs. For those cases in which in vivo transcription patterns can be duplicated, the minichromosomes will be examined for induction-correlated features. These would include: (i) the presence of regulatory proteins bound at the promoter, or at other known regulatory sites (enhancers), and (ii) changes in the configuration of the promoter (absence of nucleosomes, presence of denatured regions, etc.).