Sampath Vinaya, Balakrishnan Bindu, Verma-Gaur Jiyoti, Onesti Silvia, Sadhale Parag P
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
J Biol Chem. 2008 Feb 15;283(7):3923-31. doi: 10.1074/jbc.M708746200. Epub 2007 Dec 4.
Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4.Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.
真核生物RNA聚合酶II的两个亚基Rpb7和Rpb4形成一个亚复合物,该亚复合物在RNA聚合酶I和III中也有对应物。尽管已解析出12亚基RNA聚合酶II的中等分辨率结构,但该亚复合物与核心聚合酶之间接触区域的相对贡献以及破坏这些区域的后果尚未得到详细研究。我们在酿酒酵母Rpb7的N端核糖核蛋白样结构域中鉴定出了一些突变,这些突变影响其在某些应激反应中的作用,如在高温下生长和孢子形成。这些突变增加了Rpb7对Rpb4的依赖性,以便与聚合酶的其他部分相互作用。互补分析和RNA聚合酶下拉试验表明,Rpb4.Rbp7亚复合物通过两个关键的相互作用点与核心RNA聚合酶II的其他部分结合:一个在Rpb7的N端核糖核蛋白样结构域,另一个在Rpb4部分有序的N端区域。这些发现与12亚基聚合酶的晶体结构一致。我们在此表明,晶体结构中预测的Rpb4 N端区域与Rpb2的弱相互作用实际上在体内亚复合物与核心的相互作用中起重要作用。我们的突变分析还表明,Rpb7通过其与聚合酶其他部分相互作用的能力在细胞中发挥重要作用。