Analysis of regulation mechanisms of the fission yeast RNA polymerase II

RNA聚合酶II（Pol II）最显著的调控机制是在转录周期中Rpb 1亚基C端结构域（CTD）的可逆磷酸化。CTD-磷酸酶Fcp 1/通用转录因子TFIIF/Pol II的复合物也含有Tfg 3，由首席研究员和同事先前从粟酒裂殖酵母中分离，并且Rpb 4亚基被鉴定为Pol II上的Fcp 1结合位点。本课题主要研究结果如下：1. Tfg 3的特性分析。（1）粟酒裂殖酵母Tfg 3是TFIIF亚基。（2）Tfg 3也是TFIID的一个亚基，Tfg 3与TFIID的结合在体内和体外都具有温度依赖性。（3）粟酒裂殖酵母tfg 3 ^-菌株对多种胁迫敏感。（4）分离出tfg 3 ^-突变的多拷贝抑制基因。（5）Tfg 3在高温下以基因特异性方式发挥功能。（1）Rpb 4的7个预测螺旋中的第2和第3个对于Rpb 4组装到Pol II是必需的。（2）第一螺旋的缺失导致细胞的温度敏感性和CTD对未结合的Pol II复合物的过度磷酸化，表明Rpb 4对Fcp 1具有调节作用。（3）第4 ~ 7个螺旋的缺失也引起了细胞的温度敏感性。在细胞中，在允许的温度下，更多的Fcp 1与接合的Pol II复合物结合，并且CTD变得低磷酸化。3.以下蛋白质被生化鉴定为Rpb 4/Rpb 7结合蛋白，并提出了Pol II的新调节机制。（1）甘油醛-3-磷酸脱氢酶（报道为哺乳动物转录介质复合物的组分）。（2）肌动蛋白（报道为染色质重塑因子复合物的组分）。

The most remarkable regulation mechanism of RNA polymerase II (Pol II) is reversible phosphorylation of the C-terminal domain (CTD) of Rpb1 subunit during a transcription cycle. A complex of CTD-phosphatase Fcp1/general transcription factor TFIIF/Pol II, which also contains Tfg3, was isolated from Schzosaccharomyces pombe by the head investigator and coworkers previously, and the Rpb4 subunit was identified as the Fcp1 binding site on Pol II. In this project, the following results were obtained.1.Characterization of Tfg3 revealed the following.(1)S.pombe Tfg3 is a TFIIF subunit.(2)Tfg3 is a subunit of TFIID as well, and the Tfg3 association to TFIID is temperature dependent in vivo and in vitro.(3)S.pombe tfg3^- strains are sensitive to a variety of stresses.(4)Multi copy suppressor genes of the tfg3^- mutation were isolated.(5)Tfg3 functions in a gene specific manner at higher temperature.2.Structure-function analysis of Rpb4 subunit of Pol II revealed the following.(1)The 2nd and 3rd of 7 predicted helices of Rpb4 are essential for the Rpb4 assembly to Pol II.(2)Deletion of the 1st helix caused thermo-sensitivity to the cell and CTD hyper-phosphorylation to the unengaged Pol II complex, suggesting a regulatory role of Rpb4 on Fcp1.(3)Deletion of the 4th to 7th helices also caused thermo-sensitivity to the cell. In the cell, more Fcp1 binds to engaged Pol II complex at permissive temperature and the CTD becomes hypo-phosphorylated.3.The following proteins were identified biochemically as Rpb4/Rpb7 binding proteins and novel regulation mechanisms of Pol II were suggested.(1)Glyceraldehyde-3-phosphate dehydrogenase (reported as a component of mammalian transcription mediator complex).(2)Actin (reported as a component of chromatin remodeling factor complex).