Mutational analysis of yeast TFIIB. A functional relationship between Ssu72 and Sub1/Tsp1 defined by allele-specific interactions with TFIIB.
Mutational analysis of yeast TFIIB. A functional relationship between Ssu72 and Sub1/Tsp1 defined by allele-specific interactions with TFIIB.
复制标题
酵母 TFIIB 的突变分析。
DOI：
10.1093/genetics/153.2.643
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发表时间：
1999
期刊：
影响因子：
3.3
通讯作者：
Hampsey,M
中科院分区：
文献类型：
--
作者：
Wu,WH;Pinto,I;Chen,BS;Hampsey,M
TFIIB是RNA聚合酶II核心转录机制的重要组成部分。先前的研究已经定义了与其他转录因子相互作用和体外基础转录所需的TFIIB结构域。在本文报道的研究中，我们研究了TFIIB在体内转录起始的结构要求。通过易错聚合酶链反应产生编码改变形式的酵母TFIIB的sua 7突变的文库，并筛选条件性生长缺陷。定义并表征了TFIIB中的22个单氨基酸置换。这些替换分布在整个蛋白质中，主要发生在遗传学保守的位置。大多数替代对稳态蛋白水平几乎没有影响，这意味着每种替代都影响TFIIB功能，而不是合成或稳定性。与最初的sua 7突变体相反，除了一个例外，所有的替换都对起始位点选择没有影响，这表明特定的TFIIB结构缺陷影响转录的准确性。sua 7等位基因的集合，包括最初的sua 7等位基因，用于研究ssu 72和sub1之间相互作用的等位基因特异性，这两者最初被鉴定为sua 7突变的抑制子（SUB1 2μ）或增强子（sub1Δ，ssu 72 -1）。我们发现，ssu 72 -1和sub1Δ与sua 7的相互作用是等位基因特异性的; ssu 72和sub1的等位基因特异性重叠;与ssu 72和sub1相互作用的每个sua 7等位基因影响转录起始位点选择的准确性。这些结果证明了TFIIB，Ssu 72和Sub1之间的功能相互作用，并表明这些相互作用在RNA聚合酶II的起始位点选择机制中发挥作用。
TFIIB is an essential component of the RNA polymerase II core transcriptional machinery. Previous studies have defined TFIIB domains required for interaction with other transcription factors and for basal transcription in vitro. In the study reported here we investigated the TFIIB structural requirements for transcription initiation in vivo. A library of sua7 mutations encoding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects. Twenty-two single amino acid replacements in TFIIB were defined and characterized. These replacements are distributed throughout the protein and occur primarily at phylogenetically conserved positions. Most replacements have little or no effect on the steady-state protein levels, implying that each affects TFIIB function rather than synthesis or stability. In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start site selection, indicating that specific TFIIB structural defects affect transcriptional accuracy. This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactions between ssu72 and sub1, both of which were initially identified as either suppressors (SUB1 2μ) or enhancers (sub1Δ, ssu72-1) of sua7 mutations. We show that the interactions of ssu72-1 and sub1Δ with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; and that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection. These results demonstrate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the mechanism of start site selection by RNA polymerase II.