Wu J, Awrey D E, Edwards A M, Archambault J, Friesen J D
Banting and Best Department of Medical Research, University of Toronto, ON, Canada.
Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11552-7. doi: 10.1073/pnas.93.21.11552.
We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.
我们之前报道过酵母RNA聚合酶II（RNAPII）最大亚基编码基因突变的分离及遗传特征分析，这些突变导致对6-氮尿嘧啶（6AU）敏感的生长特性。有人提出这些突变影响了RNAPII与转录延伸因子TFIIS之间的功能相互作用，因为突变菌株的6AU敏感表型与TFIIS产生缺陷的菌株相似，并且可以通过增加酵母TFIIS编码基因PPR2的剂量来抑制。从两个独立的6AU敏感酵母突变体和野生型（wt）细胞中纯化并鉴定了RNAPII。在体外，在没有TFIIS的情况下，纯化的野生型聚合酶和两种突变型聚合酶在聚合、在内在转录终止位点的通读以及新生RNA切割方面表现出相似的比活性。与野生型聚合酶不同，在非依赖启动子的转录、通读或切割实验中，添加3倍摩尔过量的TFIIS均不能刺激两种突变型聚合酶。然而，在TFIIS与RNAPII的摩尔比大于600:1时，观察到突变型RNAPII切割新生RNA和通读内在终止位点的能力受到刺激。与这些发现一致，发现突变型聚合酶对TFIIS的结合亲和力与野生型酶相比降低了50倍以上。这些研究表明TFIIS在酵母RNAPII转录调控中起重要作用，并确定了TFIIS在RNAPII上可能的结合位点。
