Abstract：
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
摘要：
RNA聚合酶II(RNAPolII)介导的转录,蛋白质复合物在不同步骤的高度调节过程。这里,研究RNAPolII和转录因子结合和解离动力学，我们使用CRISPR-Cas9产生了内源性光活化GFP（PA-GFP）和HaloTag敲击素，使我们能够跟踪果蝇多烯染色体中诱导的Hsp70位点的分子群体。我们发现在热休克反应的早期,小RNAPolII和DRB敏感性诱导因子（DSIF）被重新用于迭代循环的转录。令人惊讶的是,尽管通过染色质免疫沉淀（ChIP）测定在整个基因体中发现了PAF1和Spt6，它们表现出明显不同的结合行为。此外,我们发现PAF1和Spt6仅在正转录延伸因子（P-TEFb）介导的磷酸化和RNAPolII启动子近端暂停逃逸后募集。最后,我们观察到PAF1对于核小体密度低的高表达基因的转录可能是消耗性的。因此，我们的活细胞成像数据为转录调控的机制模型提供了关键约束.
