Jackson B M, Drysdale C M, Natarajan K, Hinnebusch A G
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 1996 Oct;16(10):5557-71. doi: 10.1128/MCB.16.10.5557.
GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. The N-terminal 100 amino acids of GCN4 contains a potent activation function that confers high-level transcription in the absence of the centrally located acidic activation domain (CAAD) delineated in previous studies. To identify specific amino acids important for activation by the N-terminal domain, we mutagenized a GCN4 allele lacking the CAAD and screened alleles in vivo for reduced expression of the HIS3 gene. We found four pairs of closely spaced phenylalanines and a leucine residue distributed throughout the N-terminal 100 residues of GCN4 that are required for high-level activation in the absence of the CAAD. Trp, Leu, and Tyr were highly functional substitutions for the Phe residue at position 45. Combined with our previous findings, these results indicate that GCN4 contains seven clusters of aromatic or bulky hydrophobic residues which make important contributions to transcriptional activation at HIS3. None of the seven hydrophobic clusters is essential for activation by full-length GCN4, and the critical residues in two or three clusters must be mutated simultaneously to observe a substantial reduction in GCN4 function. Numerous combinations of four or five intact clusters conferred high-level transcription of HIS3. We propose that many of the hydrophobic clusters in GCN4 act independently of one another to provide redundant means of stimulating transcription and that the functional contributions of these different segments are cumulative at the HIS3 promoter. On the basis of the primacy of bulky hydrophobic residues throughout the activation domain, we suggest that GCN4 contains multiple sites that mediate hydrophobic contacts with one or more components of the transcription initiation machinery.
GCN4是碱性亮氨酸拉链（bZIP）家族中的一种转录激活因子，可调控酿酒酵母中氨基酸生物合成基因。GCN4的N端100个氨基酸包含一种强大的激活功能，在缺乏先前研究中所描述的位于中央的酸性激活结构域（CAAD）的情况下，仍能赋予高水平转录。为了确定对N端结构域激活重要的特定氨基酸，我们对一个缺失CAAD的GCN4等位基因进行诱变，并在体内筛选导致HIS3基因表达降低的等位基因。我们发现GCN4的N端100个残基中分布着四对紧密相邻的苯丙氨酸和一个亮氨酸残基，它们是在没有CAAD时进行高水平激活所必需的。色氨酸、亮氨酸和酪氨酸是45位苯丙氨酸残基的高效功能替代物。结合我们之前的研究结果，这些结果表明GCN4包含七个芳香族或大体积疏水残基簇，它们对HIS3的转录激活有重要贡献。这七个疏水簇中没有一个对于全长GCN4的激活是必需的，并且必须同时突变两到三个簇中的关键残基才能观察到GCN4功能的显著降低。四个或五个完整簇的多种组合赋予了HIS3高水平转录。我们提出，GCN4中的许多疏水簇彼此独立发挥作用，提供冗余的转录刺激方式，并且这些不同片段的功能贡献在HIS3启动子处是累积的。基于整个激活结构域中大体积疏水残基的首要地位，我们认为GCN4包含多个位点，可介导与转录起始机制的一个或多个组分的疏水接触。
