Miyao T, Woychik N A
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Department of Molecular Genetics and Microbiology, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA.
Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15281-6. doi: 10.1073/pnas.95.26.15281.
A mutation in RPB5 (rpb5-9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts. In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart. The defects noted with rpb5-9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.
RPB5（rpb5-9）是一种组装到RNA聚合酶I、II和III中的必需RNA聚合酶亚基，其突变揭示了该亚基在转录激活中的作用。使用突变全细胞提取物进行体外转录时，GAL4-VP16介导的激活作用受到损害。使用诱导型报告质粒和Northern分析进行的体内实验支持体外数据，并表明RPB5在一些但不是所有启动子处影响激活。值得注意的是，该突变定位到人类RPB5的一个保守区域，其他人认为该区域在激活中起作用。包含该保守区域的嵌合人-酵母RPB5现在可以替代其酵母对应物发挥功能。rpb5-9所显示的缺陷与在RPB1-羧基末端结构域（CTD）截短突变体中看到的缺陷相似。我们证明RPB5和RPB1-CTD在激活中具有重叠作用，因为双突变体是合成致死的，并且在GAL1/10启动子处具有加剧的激活缺陷。这些研究表明RNA聚合酶II中有多个激活靶点，并且RPB5和CTD在激活中具有相似作用。
