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Mutational studies of yeast transcription factor IIB in vivo reveal a functional surface important for gene activation

机译:体内酵母转录因子IIB的突变研究表明功能表面对于基因激活很重要

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摘要

Recent experiments in yeast (Saccharomyces cerevisiae) cells have identified a species-specific region of yeast transcription factor IIB (TFIIB) located at residues 144–157. According to the human TFIIB structure, this region is part of a solvent-exposed helix in the first repeat of the carboxyl-terminal core domain. In this report, we systematically analyze four positions in this region (Lys-147, Cys-149, Lys-151, and Glu-152) that together have been shown previously to be important for yeast TFIIB’s function in vivo. Our experiments suggest that all of these four positions, and in particular positions 151, 149, and 152, are critical for yeast TFIIB’s ability to support cell growth. In addition, we describe an intragenic suppressor screening experiment to identify mutations that reverse, or partially reverse, the temperature-sensitive phenotype of a yeast TFIIB derivative bearing amino acid changes at these four positions to human residues. The suppressor mutations reveal changes at positions 115, 117, and 182 that are located outside the species-specific region of yeast TFIIB, suggesting an extended surface available to interact with other proteins. Finally, we demonstrate that the suppressor mutations restore gene activation in vivo, further supporting the idea that one important function of yeast TFIIB in living cells is to mediate gene activation.
机译:最近在酵母(Saccharomyces cerevisiae)细胞中进行的实验已经确定了一个位于酵母菌转录因子IIB(TFIIB)的物种特异性区域,位于残基144-157。根据人TFIIB结构,该区域是羧基末端核心结构域的第一个重复序列中溶剂暴露的螺旋结构的一部分。在这份报告中,我们系统地分析了该区域的四个位置(Lys-147,Cys-149,Lys-151和Glu-152),这些位置先前已被证明对酵母TFIIB的体内功能至关重要。我们的实验表明,这四个位置,尤其是151、149和152位,对于酵母TFIIB支持细胞生长的能力至关重要。另外,我们描述了一种基因内抑制物筛选实验,以鉴定可逆转或部分逆转具有氨基酸突变的酵母TFIIB衍生物的温度敏感表型的突变,所述酵母TFIIB衍生物在这四个位置上与人残基发生氨基酸改变。抑制突变揭示了位于酵母TFIIB的物种特异性区域之外的位置115、117和182的变化,表明存在可与其他蛋白质相互作用的扩展表面。最后,我们证明了抑制突变体在体内恢复了基因激活,进一步支持了酵母TFIIB在活细胞中的一项重要功能是介导基因激活的想法。

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