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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase Ⅱ in Saccharomyces cerevisiae
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Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase Ⅱ in Saccharomyces cerevisiae

机译：酿酒酵母中的裂解/聚腺苷酸化因子IA与RNA聚合酶Ⅱ的羧基末端结构域相关

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摘要

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase Ⅱ plays an important role in transcription and process- ing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/ polyadenylation factor IA of Saccharomyes cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphory- lated CTD. This interaction is confirmed for Rna15p by yqast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.
机译：RNA聚合酶Ⅱ最大的亚基的羧基末端结构域（CTD）通过与转录和RNA加工因子相互作用，在新生转录本的转录和加工中发挥重要作用。我们在这里显示，酿酒酵母的裂解/聚腺苷酸化因子IA直接接触CTD。首先，通过酵母提取物的亲和色谱实验，我们证明了该复合物的Rna15p，Rna14p和Pcf11p亚基与磷酸化的CTD相关。 yqast两杂交分析证实了Rna15p的这种相互作用。其次，基于与重组蛋白的体外结合研究，Pcf11p而非Rna15p与磷酸化的CTD直接接触。这些发现建立了裂解/聚腺苷酸化因子IA与CTD的直接相互作用。此外，对温度敏感突变株进行的转录运行的定量分析表明，功能性Rna14p或Pcf11p的缺乏比功能性Rna15p的缺乏更严重地影响转录终止。此外，这些数据强化了CTD磷酸化充当初级转录本成熟的调节机制这一概念。

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