Muguet Alexia, Gardrat Thomas, Conconi Antonio, Paillé Audrey
Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, QC, Canada.
Methods Mol Biol. 2025;2919:133-154. doi: 10.1007/978-1-0716-4486-7_8.
In the nucleoli of eukaryotic cells, the multiple copies of ribosomal RNA genes (rRNA genes) coexist in two different forms that have distinct characteristics: transcribed (active) and non-transcribed (inactive) units. "Active" rRNA genes are loaded with RNA polymerase I and are largely depleted of nucleosomes, whereas "inactive" rRNA genes are covered with two copies of the four histone proteins that are folded in nucleosomes. A third form of chromatin is observed in Saccharomyces cerevisiae (here called as yeast) arrested in the G1 phase of the cell cycle. In yeast synchronized before DNA replication, nucleosomes are also absent in the non-transcribed rRNA genes, which are described as "open" units.The presence of two distinct groups of rRNA genes compromises the interpretation of standard biochemical assays that are employed to study the structure of chromatin during DNA transcription, DNA replication, and DNA repair. This chapter describes protocols to investigate the association of histone proteins with rRNA genes in yeast. In addition, it provides a comprehensive list of studies that applied psoralen photo-crosslinking to follow the structure of rRNA gene chromatin in a variety of high eukaryotic cells.
在真核细胞的核仁中，核糖体RNA基因（rRNA基因）的多个拷贝以两种具有不同特征的不同形式共存：转录（活性）和非转录（非活性）单元。“活性”rRNA基因负载有RNA聚合酶I，并且基本上没有核小体，而“非活性”rRNA基因则被折叠在核小体中的四种组蛋白的两个拷贝所覆盖。在细胞周期G1期停滞的酿酒酵母（此处称为酵母）中观察到第三种染色质形式。在DNA复制前同步化的酵母中，非转录的rRNA基因中也没有核小体，这些基因被描述为“开放”单元。两组不同的rRNA基因的存在影响了用于研究DNA转录、DNA复制和DNA修复过程中染色质结构的标准生化分析的解释。本章描述了研究酵母中组蛋白与rRNA基因关联的实验方案。此外，它提供了一份全面的研究列表，这些研究应用补骨脂素光交联来追踪各种高等真核细胞中rRNA基因染色质的结构。
