Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan.
PLoS One. 2007 Aug 15;2(8):e746. doi: 10.1371/journal.pone.0000746.
Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5' UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity.
Cys2His2 锌指是真核生物 DNA 结合基序，能够区分不同的 DNA 序列，适合工程设计人工转录因子。在这项工作中，我们使用酿酒酵母（Saccharomyces cerevisiae）来研究定制锌指蛋白激活 FLO11 基因表达的能力及其表型后果。鉴定出两个三指肽，它们以纳摩尔亲和力识别 FLO11 基因 5'UTR 的位点。三指结构域及其组合的六指模体识别 18 个碱基对的位点，被融合到 VP16 或 VP64 的激活结构域中。这些转录因子构建体保留了它们的 DNA 结合能力，其中六指结构域的亲和力最高。然而，当在单倍体酵母细胞中表达时，只有一种三指重组转录因子能够有效地激活 FLO11 的表达。与野生型不同，具有这种转录激活的细胞表现出侵袭性生长和生物膜形成，而不需要葡萄糖耗尽。VP16 和 VP64 结构域似乎在 FLO11 表达的激活中同样有效，在表型改变方面具有可比的效果。我们得出结论，定制转录因子在细胞中的功能活性不容易通过体外 DNA 结合活性来预测。
