Sub1 and RPA associate with RNA Polymerase II at different stages of transcription

Single stranded DNA binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) pre-initiation complexes (PICs) identified Sub1 and the Replication Protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the non-template strand of RNApII complexes during initiation and elongation, respectively. Chip-chip from wt and sub1D cells with Rfa1 Chromatin immunoprecipitation (ChIP) of Rfa1 in wt and sub1D yeast demonstrated that Rfa1 localization correlates with RNA Polymerase II and is increased at some transcription start sites when Sub1 has been deleted. Comparison of Rfa1 localization in wt vs sub1D yeast

单链DNA结合蛋白（single-stranded DNA binding protein）在核酸代谢中发挥诸多功能，但其在转录过程中的重要性仍未明确。通过对RNA聚合酶II（RNA polymerase II，RNApII）预起始复合物（pre-initiation complexes，PICs）开展定量蛋白质组学分析，研究人员鉴定出Sub1与复制蛋白A复合物（Replication Protein A complex，RPA），二者均可结合单链DNA（single-stranded DNA，ssDNA）。Sub1是哺乳动物辅激活因子PC4的同源蛋白，其与启动子解链所需的多种因子存在强烈的遗传互作。Sub1在体外可定位于转录泡附近，且在体内依赖预起始复合物组装结合至启动子区域。与之相反，RPA定位于活性基因的转录区域，其定位与正在转录的RNApII高度相关，但不依赖于复制过程。RFA1与转录延伸因子编码基因存在遗传互作。值得注意的是，在Sub1缺失或携带单链DNA结合突变体的细胞中，活性启动子处的RPA水平升高，这提示二者存在共同结合位点的竞争。我们提出，Sub1与RPA分别在转录起始与延伸阶段，与RNApII复合物的非模板链相结合。通过针对Rfa1的染色质免疫沉淀（Chromatin immunoprecipitation，ChIP）对野生型（wild-type，wt）与sub1D酵母开展ChIP-chip实验，结果显示Rfa1的定位与RNA聚合酶II存在相关性，且在Sub1缺失时，部分转录起始位点处的Rfa1信号出现增强。对野生型与sub1D酵母中Rfa1的定位进行比较