4tU RNA-seq from wild-type and Rpb7-QHF cells

Our structural and biochemical studies show that S. cerevisiae RNA Polymerase II (RNAPII) homodimerizes through the stalk domain (formed by the Rpb4-Rpb7 subunits). To explore the biological impact of disrupting this interaction we introduced a triple point mutation at the RNAPII dimerization interface in Rpb7 (Q96A, H97A, F109K). To test the impact of the dimerization mutant on RNA synthesis and 3' end processing in WT and Rpb7-QHF cells, we labelled nascent transcripts with 4tU which enabled subsequent biotinylation and purification of newly-synthesized transcripts. Nascent transcripts were then used to prepare strand-specific RNA-seq libraries.

本研究通过结构生物学与生物化学实验证实，酿酒酵母（Saccharomyces cerevisiae，S. cerevisiae）RNA聚合酶II（RNA Polymerase II，RNAPII）可通过由Rpb4-Rpb7亚基构成的茎区发生同源二聚化。为探究破坏该相互作用的生物学效应，本研究在Rpb7的RNAPII二聚化界面引入三联点突变（Q96A、H97A、F109K）。为检测该二聚化突变体对野生型（wild type，WT）及Rpb7-QHF细胞中RNA合成与3'端加工过程的影响，我们采用4tU标记新生转录本，这为后续新合成转录本的生物素标记与纯化提供了条件。随后利用上述新生转录本构建链特异性RNA测序（RNA-seq）文库。