Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, USA.
Nature. 2011 Jan 20;469(7330):368-73. doi: 10.1038/nature09652.
Recent studies of transcription have revealed a level of complexity not previously appreciated even a few years ago, both in the intricate use of post-initiation control and the mass production of rapidly degraded transcripts. Dissection of these pathways requires strategies for precisely following transcripts as they are being produced. Here we present an approach (native elongating transcript sequencing, NET-seq), based on deep sequencing of 3' ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution. Application of NET-seq in Saccharomyces cerevisiae reveals that although promoters are generally capable of divergent transcription, the Rpd3S deacetylation complex enforces strong directionality to most promoters by suppressing antisense transcript initiation. Our studies also reveal pervasive polymerase pausing and backtracking throughout the body of transcripts. Average pause density shows prominent peaks at each of the first four nucleosomes, with the peak location occurring in good agreement with in vitro biophysical measurements. Thus, nucleosome-induced pausing represents a major barrier to transcriptional elongation in vivo.
最近的转录研究揭示了即使在几年前也未被充分认识的复杂性水平，包括起始后控制的复杂使用和快速降解转录本的大量产生。这些途径的剖析需要精确跟踪转录本的策略，因为它们正在被产生。在这里，我们提出了一种方法（天然延伸转录测序，NET-seq），该方法基于与 RNA 聚合酶相关的新生转录本 3' 端的深度测序，以核苷酸分辨率监测转录。在酿酒酵母中的 NET-seq 的应用表明，尽管启动子通常能够进行发散转录，但 Rpd3S 去乙酰化复合物通过抑制反义转录本起始来对大多数启动子施加强烈的方向性。我们的研究还揭示了整个转录本中普遍存在的聚合酶暂停和回溯。平均暂停密度在每个前四个核小体上都有明显的峰值，峰值位置与体外生物物理测量非常吻合。因此，核小体诱导的暂停是体内转录延伸的主要障碍。
