Shih H M, Goldman P S, DeMaggio A J, Hollenberg S M, Goodman R H, Hoekstra M F
Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13896-901. doi: 10.1073/pnas.93.24.13896.
The Escherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selection. This assay, designated the "split-hybrid system," contains a two-component reporter. The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream of HIS3. Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression. TetR subsequently binds to the tet operators, blocking the expression of HIS3 and preventing yeast growth in media lacking histidine. The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133. Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains. The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP. These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction. The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein-protein interactions.
利用大肠杆菌四环素阻遏物（TetR）操纵子系统开发了酵母双杂交分析的一种变体，其中蛋白质-蛋白质相互作用的破坏可通过阳性筛选来鉴定。这种分析方法称为“分裂杂交系统”，包含一个双组分报告基因。第一组分在TetR基因上游含有LexA结合位点，第二组分在HIS3基因上游含有TetR操纵子结合位点。与LexA DNA结合结构域融合的一种蛋白质与与VP16激活结构域融合的第二种蛋白质相互作用会导致TetR表达。TetR随后与四环素操纵子结合，阻断HIS3的表达并阻止酵母在缺乏组氨酸的培养基中生长。通过检测CREB及其共激活因子CREB结合蛋白（CBP）的磷酸化依赖性相互作用来分析分裂杂交系统的效用。CREB和CBP通过一种依赖于Ser-133处CREB磷酸化的相互作用而结合。该磷酸化位点的突变在标准双杂交分析中会阻止酵母生长，但在分裂杂交菌株中则允许生长。分裂杂交系统用于鉴定其他破坏其与CBP结合的CREB突变。这些突变定位在CREB磷酸化位点周围，表明CBP相互作用仅需要CREB激活结构域的一小部分。酵母分裂杂交系统在鉴定破坏蛋白质-蛋白质相互作用的突变、蛋白质、肽和药物方面应该是有用的。
