Service de Biochimie et Génétique Moléculaire, CEA-Saclay, Bâtiment 144, 91191 Gif-sur-Yvette Cedex, France.
Curr Genet. 2011 Oct;57(5):327-34. doi: 10.1007/s00294-011-0350-6. Epub 2011 Jul 15.
The catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-(832)GYNQED(837), Rpc128-(764)GYDIED(769)) and highly conserved in all cellular and viral DNA-dependent RNA polymerases. A site-directed mutagenesis of this dicarboxylic motif reveals its strictly essential character in RNA polymerase III, with a slightly less stringent pattern in RNA polymerase II, where rpb2-E836Q and other substitutions completely prevent growth, whereas rpb2-E836A combines a dominant growth defect with severe lethal sectoring. A mild but systematic increase in RNA polymerase occupancy and a strict dependency on the transcript cleavage factor TFIIS (Dst1) also suggest a slower rate of translocation or higher probability of transcriptional stalling in this mutation. A conserved nucleotide triphosphate funnel domain binds the Rpb2-(832)GYNQED(837) loop by an Rpb2-R(1020)/Rpb2-D(837) salt-bridge. Molecular dynamic simulations reveal a second bridge (Rpb1-K(752)/Rpb2-E(836)), which may account for the critical role of the invariant Rpb2-E(836). Rpb2-E(836) and the funnel domain are not found among the RNA-dependent eukaryotic RNA polymerases and may thus represent a specific adaptation to double-stranded DNA templates.
酵母 RNA 聚合酶 II 和 III 的催化中心包含其二大亚基携带的酸性环（Rpb2-(832)GYNQED(837)，Rpc128-(764)GYDIED(769)），该环在所有细胞和病毒 DNA 依赖性 RNA 聚合酶中高度保守。该二羧酸基模体的定点突变揭示了其在 RNA 聚合酶 III 中的严格必需性，在 RNA 聚合酶 II 中模式稍宽松，其中 rpb2-E836Q 和其他取代完全阻止生长，而 rpb2-E836A 则与严重的致死性部分性缺陷相结合产生显性生长缺陷。聚合酶占有率的轻度但系统增加以及对转录切割因子 TFIIS（Dst1）的严格依赖性也表明，该突变中易位的速率较慢或转录停滞的可能性更高。保守的核苷酸三磷酸漏斗域通过 Rpb2-R(1020)/Rpb2-D(837)盐桥与 Rpb2-(832)GYNQED(837)环结合。分子动力学模拟揭示了第二个桥（Rpb1-K(752)/Rpb2-E(836)），这可能解释了不变的 Rpb2-E(836)的关键作用。Rpb2-E(836)和漏斗域在 RNA 依赖性真核 RNA 聚合酶中不存在，因此可能代表对双链 DNA 模板的特定适应。
