Walter Wendy, Studitsky Vasily M
Department of Biochemistry and Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Methods. 2004 May;33(1):18-24. doi: 10.1016/j.ymeth.2003.10.016.
Transcription through the nucleosome by Saccharomyces cerevisiae RNA polymerase II (Pol II) is characterized by an almost absolute block to transcription at physiological ionic strength and displacement of one H2A/H2B dimer to form a hexasome [Mol. Cell 9 (2002) 541]. In previous studies of Pol II transcription through chromatin, templates containing nucleosomes in multiple positions were used. These templates do not allow detailed analysis of the mechanism of transcription through chromatin. Here, we describe the development of a new template that is only long enough to accommodate a single nucleosome position along the DNA so that all of the templates are identical and allow for more in-depth analysis. After ligation of the nucleosome to promoter DNA or assembled elongation complexes, the mechanism of transcription through this uniquely positioned nucleosome by various RNA polymerases can be analyzed.
酿酒酵母RNA聚合酶II（Pol II）通过核小体的转录具有以下特征：在生理离子强度下转录几乎完全受阻，并且一个H2A/H2B二聚体发生位移以形成六聚体[《分子细胞》9（2002年）541]。在先前关于Pol II通过染色质进行转录的研究中，使用了在多个位置含有核小体的模板。这些模板不允许对通过染色质的转录机制进行详细分析。在此，我们描述了一种新模板的开发，该模板的长度仅足以容纳沿DNA的单个核小体位置，从而所有模板都是相同的，并允许进行更深入的分析。在将核小体连接到启动子DNA或组装延伸复合物后，可以分析各种RNA聚合酶通过这个独特定位的核小体的转录机制。
