Locus-specific isolation of transcription elongation complexes from Saccharomyces cerevisiae

Transcription of mRNA products by RNA polymerase II (Pol II) is a multi-stage event subject to a multitude of regulatory processes. Transcription, RNA processing, and chromatin related factors all interact with Pol II to ensure proper timing and coordination of transcription and co-transcriptional processes. Many regulators must function simultaneously to coordinate these processes, yet few strategies exist to explore the full complement of factors regulating specific stages of transcription. To this end we developed a strategy to purify Pol II elongation complexes from specific loci of a single gene, namely the 5′ and 3′ regions, using sequences in the nascent RNA. Applying this strategy to Saccharomyces cerevisiae we determined the specific set of factors that interact with Pol II at precise stages during transcription. We identify many known region-specific factors as well as determine a role for the transcription termination factor Rai1 in regulating the early stages of transcription genome-wide. We also demonstrate a role for the ubiquitin ligase Bre1 in regulating Pol II dynamics during the latter stages of transcription. This strategy for gene and loci-specific isolation of transcription complexes will provide a useful tool to explore the host of factors that regulate the different stages of transcription and coordinate co-transcriptional processes.RNA聚合酶II（RNA polymerase II，Pol II）介导的mRNA产物转录是一个多阶段过程，受到多种调控机制的精准调控。转录、RNA加工与染色质相关因子均与Pol II发生相互作用，以保障转录及共转录过程的时序正确与协同有序。诸多调控因子需协同发挥作用以协调上述过程，但目前鲜有策略能够全面探究调控转录特定阶段的全部因子集合。为此，我们开发了一种策略，可借助新生RNA中的序列，从单个基因的特定位点（即5'端与3'端区域）纯化Pol II延伸复合物。我们将该策略应用于酿酒酵母（Saccharomyces cerevisiae），确定了转录过程中不同精确阶段与Pol II相互作用的特定因子集合。我们不仅鉴定出多种已知的区域特异性因子，还明确了转录终止因子Rai1在全基因组范围内调控转录早期阶段的功能。此外，我们还证实了泛素连接酶Bre1在转录后期调控Pol II动态变化的功能。这种可实现基因与位点特异性分离转录复合物的策略，将为探究调控转录不同阶段并协调共转录过程的各类因子提供一款实用工具。