A NOVEL GENETIC SYSTEM TO DETECT PROTEIN PROTEIN INTERACTIONS
A NOVEL GENETIC SYSTEM TO DETECT PROTEIN PROTEIN INTERACTIONS
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DOI：
10.1038/340245a0
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发表时间：
1989-07-20
期刊：
影响因子：
64.8
通讯作者：
SONG, OK
中科院分区：
文献类型：
--
作者：
FIELDS, S;SONG, OK
通常使用生物化学技术，如交联法、免疫共沉淀法和层析共分级法来研究两种蛋白质之间的相互作用。我们利用酿酒酵母的GAL4蛋白的特性，建立了一个新的遗传系统来研究这些相互作用。该蛋白是编码半乳糖利用酶的基因表达所需的转录激活剂。它由两个可分离的功能必需结构域组成：一个N-末端结构域与特定的DNA序列(UASG)结合；一个C-末端结构域包含激活转录所必需的酸性区域2，3。我们已经建立了一个包含部分GAL4的两个杂交蛋白系统：GAL4 DNA结合域融合到蛋白X，GAL4激活区融合到蛋白质Y。如果X和Y能够形成蛋白质-蛋白质复合体，并重建GAL4结构域的邻近，则发生由UASG调控的基因的转录。我们已经使用了两种已知相互作用的酵母蛋白-SNF1和SNF4来测试这个系统。只有当两个杂交体都存在于一个细胞中时，才能获得高转录活性。该系统可用作通过使用简单的半乳糖选择来鉴定与已知蛋白质相互作用的蛋白质的通用方法。
PROTEIN-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeastSaccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization1. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription2,3. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, tran-scription of a gene regulated by UASGoccurs. We have tested this system using two yeast proteins that are known to interact—SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.