Cys2His2 鋅指結構域（domain）乃真核生物蛋白質中最常見的去氧核醣核酸結合基序（ motif），可辨識多種不同組合的去氧核醣核酸序列。鋅指蛋白模組（ module）因其結構的穩定性可於蛋白質工程中做為多元變異的台架。轉錄因子 Zif268 的三指模組已被用於構築由噬菌體呈現之組合基因庫的框架來闡釋鋅指 DNA 相互辨識的理論。近年來研究者開發對鋅指選擇及設計的方法已可構築幾乎能標的於雙螺旋 DNA 任何位置的鋅指蛋白質。然而以鋅指蛋白質為基礎由体外選擇及設計的人工重組轉錄因子與其在生物體內活性的關係仍無完整有系統的分析。本計劃的目的是利用出芽酵母菌為模式系統研究量身設計訂做的鋅指蛋白質對引導控制基因表達的能力。我探討分屬兩個不同裂殖原活化蛋白質激酵素路徑而涉及高張壓甘油反應和侵略性生長的基因。我將建構量身訂做的轉錄因子以活化下述基因之表達。其一為磷酸三甘油去氫酵素（ GPD1）基因使得剔除 HOG1 基因的出芽酵母菌能於高張壓條件下仍能生長。另一為 musin-like （MUC1）基因使單倍體的出芽酵母菌在未缺乏氮養分來源時能呈侵略性生長。為了達到能識別各種組合的 DNA 序列，我採取一種新穎的同時並行（ bipartite）選擇方法同時以兩個互補基因庫選擇兩個 DNA 結合結構域，再經重組產生能識別一九個鹼基對的三指胜。已篩選出對 MUC1 或 GPD1 啟動子序列有專一性的鋅指結合基序，三指胜。其中一個三指胜被聯結成能識別一複合十八個鹼基對的六指胜。目前完成鋅指結合基序與激活結構域 VP16 或 VP64 的粘接於載體的構築，且已將鋅指結合基序與激活結構域轉錄因子構築於能在酵母菌並可調節表達蛋白質的載體。重組轉錄因子在酵母 菌細胞內對 GPD1 或 MUC1 轉錄活化及表現型的影響將被檢視。 此研究的完成會成就一個模式系統－能深入且仔細分析依據不同 DNA 結合力的鋅指和不同功能結構域構築成的轉錄因子且在不同蛋白表達量下與生物體內基因調節之生物功能性的關係。這是以建構的鋅指蛋白質為基因開關來了解基因行為和分子醫學中臨床研應用的一大步。
Cys2His2 zinc finger domain represents the most common eukaryotic DNA binding motif capable of recognizing an assorted set of DNA sequences. The structural stability of zinc finger motif makes it an exceptionally adaptable scaffold for protein engineering. The three-finger module from the transcription factor Zif268 has been used as a framework to construct combinatorial libraries displayed on bacteriophage to elucidate the principles of zinc finger-DNA recognition. Recent progress in selection and design of strategies for zinc fingers has allowed construction of zinc finger proteins targeting at nearly any desired site on double-stranded DNA. Nevertheless, the in vivo biological activity of artificial recombinant transcription factors-based on zinc fingers in relation to their in vitro selection and design remains to be investigated systematically. I have used budding yeast Saccharomyces cerevisiae as a model system to study the ability of tailor-made zinc finger proteins in regulation of gene expression. I exploited two gene mucin-like (MUC1) gene to promote invasive growth in haploid cells without depletion of nitrogen source. To accomplish comprehensive DNA recognition, I have adopted a novel approach of bipartite selection where two complementary libraries are used in parallel to select two DNA-binding domains, and whose products are recombined to produce a three-finger peptide that recognizes a 9two of which have been linked to generate a six-finger peptide that recognize a composite 18-bp sequence. These zinc s each of which codes for protein that is target in one of the two mitogen-activated protein kinase pathways, involved in either high-osmolarity glycerol response (HOG) or invasive growth. I aim to engineer tailor-made transcription factors that activate the expression of either glycerol-3-phosphate dehydrogenase (GPD1) gene to restore growth of cells deleted in HOG1 in condition of high osmolarity or finger motifs have been attach to the activation domain VP16 from the herpes simplex virus or its derivative VP64 and have been constructed into a repressible yeast expression vector. This will allow me to examine the effects of the resulting recombinant transcription factors in yeast cells on transcriptional activation of GDP1 or MUC1 and accompanying phenotypic alterations-bp site. Several three-finger peptides recognizing promoter sequence of either MUC1 or GPD1 gene have been identified,. Completion of this study will result in a model system that permits in depth and detailed analysis of biological functionality for gene regulation in vivo in relation to differently modified functional domains by engineering zinc finger-based transcription factors. It is a major step forward in the use of engineered zinc finger proteins as gene switches in the understanding of gene behavior and in clinical applications in molecular medicine.
