Maillet I, Buhler J M, Sentenac A, Labarre J
Service de Biochimie et Génétique Moléculaire, CEA-Saclay, F-91191 Gif-sur-Yvette Cedex, France.
J Biol Chem. 1999 Aug 6;274(32):22586-90. doi: 10.1074/jbc.274.32.22586.
Rpb4p and Rpb7p are two subunits of the yeast RNA polymerase II, which form a subcomplex that can dissociate from the enzyme in vitro. Whereas RPB7 is essential, RPB4 is dispensable for cellular viability. However, the rpb4 null mutant is heat-sensitive, and it has been suggested that Rpb4p is an essential component for cellular stress response. To examine this hypothesis, we used two-dimensional gel electrophoresis to analyze the protein expression pattern of the rpb4 null mutant in response to heat shock, oxidative stress, osmotic stress, and in the post-diauxic phase. We show that this mutant is not impaired in stress induced transcriptional activation: the absence of heat shock response of the mutant is due to a general defect in RNA polymerase II activity at high temperature. Under this condition, Rpb4p is necessary to maintain the polymerase activity in vivo. The heat growth defect of the rpb4 null mutant can be partially suppressed by overexpression of RPB7, suggesting that Rpb4p maintains or stabilizes Rpb7p in the RNA polymerase. We also demonstrate that rpb4 null mutant is an appropriate tool to analyze the involvement of transcriptional events in the survival and adaptation to heat shock or other stresses.
Rpb4p和Rpb7p是酵母RNA聚合酶II的两个亚基，它们形成一个亚复合物，该亚复合物在体外可从该酶上解离。虽然RPB7是必需的，但RPB4对于细胞活力而言是可有可无的。然而，rpb4基因缺失突变体对热敏感，有人提出Rpb4p是细胞应激反应的一个必需成分。为了验证这一假设，我们使用二维凝胶电泳来分析rpb4基因缺失突变体在热休克、氧化应激、渗透应激以及在二次生长后期的蛋白质表达模式。我们发现该突变体在应激诱导的转录激活方面没有受损：突变体缺乏热休克反应是由于高温下RNA聚合酶II活性存在普遍缺陷。在这种情况下，Rpb4p对于在体内维持聚合酶活性是必需的。rpb4基因缺失突变体的热生长缺陷可通过过量表达RPB7而部分得到抑制，这表明Rpb4p在RNA聚合酶中维持或稳定Rpb7p。我们还证明，rpb4基因缺失突变体是分析转录事件在热休克或其他应激的存活和适应过程中所起作用的合适工具。
