Ha N, Hellauer K, Turcotte B
Department of Medicine, McGill University, Royal Victoria Hospital, Montreal Quebec, Canada.
Nucleic Acids Res. 1996 Apr 15;24(8):1453-9. doi: 10.1093/nar/24.8.1453.
The yeast zinc cluster protein HAP1, a member of the GAL4 family, is a transcriptional activator that binds as a homodimer to target DNA sequences. These targets include the upstream activating sequences of the CYC1 and CYC7 genes, which have no obvious sequence similarity. Even though both sites have the same affinity for HAP1, activation differs at these two sites, even when the sequences are placed in an identical promoter context. In addition, mutants of HAP1 that can bind to both sites but are specifically transcriptionally inactive at CYC7 have been previously isolated. In order to identify nucleotides that are responsible for this differential activity, we have performed random and site-directed mutagenesis of these target sites and assayed their binding to HAP1 in vitro and their activity in vivo in reporter plasmids. Our results show that HAP1 binding sites are degenerate forms of the direct repeat CGG N3 TA N CGG N3 TA. Moreover, we show that activity of HAP1 mutants defective for activation of the CYC7gene is restored by specific mutations in the CYC7 binding site. Conversely, other mutations of the target sites prevent activation by HAP1, without interfering with DNA binding. The results suggest that the sequence of the target sites influences the conformation and, hence, the activity of DNA-bound HAP1.
酵母锌簇蛋白HAP1是GAL4家族的成员，是一种转录激活因子，以同源二聚体形式与靶DNA序列结合。这些靶标包括CYC1和CYC7基因的上游激活序列，它们没有明显的序列相似性。尽管这两个位点对HAP1具有相同的亲和力，但即使将序列置于相同的启动子环境中，这两个位点的激活情况仍有所不同。此外，之前已分离出能与这两个位点结合但在CYC7处特异性转录失活的HAP1突变体。为了确定导致这种差异活性的核苷酸，我们对这些靶位点进行了随机诱变和定点诱变，并检测了它们在体外与HAP1的结合情况以及在报告质粒中在体内的活性。我们的结果表明，HAP1结合位点是直接重复序列CGG N3 TA N CGG N3 TA的简并形式。此外，我们表明，CYC7结合位点的特定突变可恢复对CYC7基因激活有缺陷的HAP1突变体的活性。相反，靶位点的其他突变会阻止HAP1的激活，而不影响DNA结合。结果表明，靶位点的序列会影响构象，进而影响与DNA结合的HAP1的活性。
