Jordán-Pla Antonio, Gupta Ishaan, de Miguel-Jiménez Lola, Steinmetz Lars M, Chávez Sebastián, Pelechano Vicent, Pérez-Ortín José E
Departamento de Bioquímica y Biología Molecular and ERI Biotecmed, Facultad de Biológicas, Universitat de València, C/Dr. Moliner 50, E46100 Burjassot, Spain.
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Nucleic Acids Res. 2015 Jan;43(2):787-802. doi: 10.1093/nar/gku1349. Epub 2014 Dec 29.
The particular behaviour of eukaryotic RNA polymerases along different gene regions and amongst distinct gene functional groups is not totally understood. To cast light onto the alternative active or backtracking states of RNA polymerase II, we have quantitatively mapped active RNA polymerases at a high resolution following a new biotin-based genomic run-on (BioGRO) technique. Compared with conventional profiling with chromatin immunoprecipitation, the analysis of the BioGRO profiles in Saccharomyces cerevisiae shows that RNA polymerase II has unique activity profiles at both gene ends, which are highly dependent on positioned nucleosomes. This is the first demonstration of the in vivo influence of positioned nucleosomes on transcription elongation. The particular features at the 5' end and around the polyadenylation site indicate that this polymerase undergoes extensive specific-activity regulation in the initial and final transcription elongation phases. The genes encoding for ribosomal proteins show distinctive features at both ends. BioGRO also provides the first nascentome analysis for RNA polymerase III, which indicates that transcription of tRNA genes is poorly regulated at the individual copy level. The present study provides a novel perspective of the transcription cycle that incorporates inactivation/reactivation as an important aspect of RNA polymerase dynamics.
真核生物RNA聚合酶在不同基因区域以及不同基因功能组中的特定行为尚未完全明确。为了深入了解RNA聚合酶II的替代活性或回溯状态，我们采用了一种基于生物素的新型基因组延伸转录（BioGRO）技术，以高分辨率对活性RNA聚合酶进行了定量定位。与传统的染色质免疫沉淀分析方法相比，对酿酒酵母中BioGRO图谱的分析表明，RNA聚合酶II在基因两端具有独特的活性图谱，这高度依赖于定位核小体。这是首次证明定位核小体对转录延伸的体内影响。5'端和聚腺苷酸化位点附近的特定特征表明，这种聚合酶在转录延伸的初始和最终阶段经历了广泛的比活性调节。编码核糖体蛋白的基因在两端呈现出独特的特征。BioGRO还首次对RNA聚合酶III进行了新生转录本分析，这表明tRNA基因在单个拷贝水平上的转录调控较差。本研究为转录周期提供了一个新的视角，将失活/重新激活纳入RNA聚合酶动力学的一个重要方面。
