Archambault J, Lacroute F, Ruet A, Friesen J D
Department of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada.
Mol Cell Biol. 1992 Sep;12(9):4142-52. doi: 10.1128/mcb.12.9.4142-4152.1992.
Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.
关于RNA聚合酶II（RNAPII）中参与转录延伸过程以及与延伸因子相互作用的区域，人们了解甚少。一种延伸因子TFIIS，通过在体外结合RNAPII并促进其通过内在暂停位点来刺激转录延伸。在酿酒酵母中，TFIIS由PPR2基因编码。从酵母基因组中删除PPR2不会致死，但会使细胞对尿嘧啶类似物6-氮尿嘧啶（6AU）敏感。在此，我们表明在编码酿酒酵母RNAPII最大亚基（RPO21）的基因中也能分离出赋予6AU敏感性的突变。一项针对赋予6AU敏感性的RPO21突变的筛选，鉴定出了7个由接头插入或随机化学诱变产生的突变。所有这7个突变改变都聚集在最大亚基的一个区域内，该区域在真核生物RNAPII中是保守的。7个rpo21突变体中有6个在高温下无法生长这一发现，突出了该区域对RNAPII功能和/或结构完整性的重要性。我们发现，增加野生型PPR2基因的剂量可以抑制rpo21突变体对6AU的敏感性，这可能是TFIIS过表达的结果。这些结果与以下推测一致：在rpo21突变体中，RNAPII-TFIIS复合物的形成是突变酶通过暂停位点的限速步骤。除了表明RNAPII最大亚基的一个区域参与转录延伸过程外，我们的观察结果还提供了体内证据，证明TFIIS参与了RNAPII的转录过程。
