Shi X, Chang M, Wolf A J, Chang C H, Frazer-Abel A A, Wade P A, Burton Z F, Jaehning J A
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262, USA.
Mol Cell Biol. 1997 Mar;17(3):1160-9. doi: 10.1128/MCB.17.3.1160.
The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase (GST)-tagged forms of CDC73, PAF1, and TFG2 functionally replacing the chromosomal copies of the genes. Isolation of GST-tagged Cdc73p and Paf1p complexes has revealed a unique form of RNA polymerase II that contains both Cdc73p and Paf1p but lacks the Srbps found in the holoenzyme. The Cdc73p-Paf1p-RNA polymerase II-containing complex also includes Gal11p, and the general initiation factors TFIIB and TFIIF, but lacks TBP, TFIIH, and transcription elongation factor TFIIS as well as the Srbps. The Srbp-containing holoenzyme does not include either Paf1p or Cdc73p, demonstrating that these two forms of RNA polymerase II are distinct. In confirmation of the hypothesis that the two forms coexist in yeast cells, we found that a TFIIF-containing complex isolated via the GST-tagged Tfg2p construct contains both (i) the Srbps and (ii) Cdc73p and Paf1p. The Srbps and Cdc73p-Paf1p therefore appear to define two complexes with partially redundant, essential functions in the yeast cell. Using the technique of differential display, we have identified several genes whose transcripts require Cdc73p and/or Paf1p for normal levels of expression. Our analysis suggests that there are multiple RNA polymerase II-containing complexes involved in the expression of different classes of protein-coding genes.
酵母CDC73和PAF1基因的产物最初被鉴定为与RNA聚合酶II相关的蛋白质。Paf1p是一种核蛋白，对酵母基因子集的细胞生长和转录调控很重要。在本研究中，我们证明CDC73的产物是一种核蛋白，在体外可直接与纯化的RNA聚合酶II相互作用。缺失CDC73会导致温度敏感型表型。将cdc73突变与更严重的paf1突变相结合不会导致增强的表型，这表明这两种蛋白质可能在相同的细胞过程中发挥作用。为了确定Cdc73p和Paf1p之间的关系以及最近描述的RNA聚合酶II全酶形式，我们构建了含有谷胱甘肽S-转移酶（GST）标记形式的CDC73、PAF1和TFG2的酵母菌株，它们在功能上取代了基因的染色体拷贝。分离带有GST标签的Cdc73p和Paf1p复合物揭示了一种独特形式的RNA聚合酶II，它包含Cdc73p和Paf1p，但缺少全酶中发现的Srbps。含有Cdc73p-Paf1p-RNA聚合酶II的复合物还包括Gal11p以及一般起始因子TFIIB和TFIIF，但缺少TBP、TFIIH和转录延伸因子TFIIS以及Srbps。含有Srbp的全酶不包括Paf1p或Cdc73p，这表明这两种形式的RNA聚合酶II是不同的。为了证实这两种形式在酵母细胞中共存的假设，我们发现通过带有GST标签的Tfg2p构建体分离的含有TFIIF的复合物同时包含（i）Srbps和（ii）Cdc73p和Paf1p。因此，Srbps和Cdc73p-Paf1p似乎定义了酵母细胞中具有部分冗余、基本功能的两种复合物。使用差异显示技术，我们鉴定了几个基因，其转录本的正常表达水平需要Cdc73p和/或Paf1p。我们的分析表明，有多种含有RNA聚合酶II的复合物参与不同类别的蛋白质编码基因的表达。
