TRANSCRIPTION OF CHROMATIN TEMPLATES

为了揭示真核细胞中的基因调控途径，有必要对RNA合成的基本机制有更深入的了解。特别是，现在人们认识到，转录本延长的控制是基因表达调控的一个重要方面。细胞核中转录的DNA模板被包装成核小体。然而，在体外转录实验中，核小体通常是转录延长的很强障碍。我们的长期目标是获得一个更准确地反映体内事件的体外转录系统。因此，有必要更全面地描述RNA聚合酶II转录核小体模板的分子机制。我们将以有限数量的例子为指导，在这些例子中，核小体DNA的一些转录已经在体外实现。例如，单核小体模板的结果强调了与核小体相邻的DNA作为核小体DNA结合面的受体的潜在重要性，当RNA聚合酶获得模板DNA时，核小体DNA结合面没有被占据。因此，我们将制备由特定位置的核小体阵列组成的多核小体模板。这将使我们能够系统地探索聚合酶-核小体和核小体-核小体间隔物在转录延长效率中的重要性。有相当多的证据表明，组蛋白N-末端结构域的修饰在基因表达控制中是重要的。我们将在这一发现和我们自己的初步结果的基础上，探索组蛋白N末端结构域的修饰对RNA聚合酶转录核小体模板能力的影响。最后，最近的工作已经确定了一种新的转录因子，FACT，它专门促进通过核小体的转录延长。我们将研究FACT的作用机制，我们还将寻找其他延伸因子对核小体模板转录的影响。

In order to unravel gene regulatory pathways in eukaryotic cells, it is necessary to achieve a much greater understanding of the basic RNA synthesis machinery. In particular, it is now appreciated that control of transcript elongation is an important aspect of the regulation of gene expression. The DNA template for transcription in the cell nucleus is packaged into nucleosomes. However, during in vitro transcription experiments nucleosomes are usually very strong barriers to transcript elongation. Our long-term goal is to obtain an in vitro transcription system that more accurately reflects in vivo events. It is therefore necessary to much more fully characterize the molecular mechanisms through which RNA polymerase II transcribes nucleosomal templates. We will be guided by the limited number of examples in which some transcription of nucleosomal DNA has been achieved in vitro. For example, results with mononucleosomal templates have emphasized the potential importance of DNA adjacent to the nucleosome as an acceptor for nucleosomal DNA binding surfaces left unoccupied as RNA polymerase acquires the template DNA. We will therefore prepare polynucleosomal templates consisting of arrays of nucleosomes in defined locations. This will allow us to systematically explore the importance of polymerase-nucleosome and nucleosome-nucleosome spacers in the efficiency of transcript elongation. There is considerable evidence that modification of the N-terminal domains of histones is important in the control of gene expression. We will build on this finding, and on our own preliminary results, to explore the effect of modifications in the histone N-terminal domains on the ability of RNA polymerase to transcribe nucleosomal templates. Finally, recent work has identified a novel transcription factor, FACT, which specifically facilitates transcript elongation through nucleosomes. We will investigate the mechanism by which FACT functions, and we will also search for effects of other elongation factors on the transcription of nucleosomal templates.