Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain
Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain
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DOI：
10.1101/gad.873901
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发表时间：
2001-04-15
影响因子：
10.5
通讯作者：
Wagner, G
中科院分区：
文献类型：
--
作者：
Hidalgo, P;Ansari, AZ;Wagner, G
GAL 4二聚化结构域（GAL 4-dd）在携带GAL 11蛋白突变体（称为GAL 11 P）的细胞中与DNA连接时是一种强大的转录激活因子。GAL 11 P（类似于GAL 11）是RNA聚合酶II全酶的一种组分。GAL 4-dd的核磁共振（NMR）研究揭示了具有C-2对称性的伸长的二聚体结构，其含有通过卷曲螺旋接触介导二聚化的三个螺旋。三个盘绕的线圈之间的两个环形成移动的凸起，引起螺旋对之间的扭转角的变化。化学位移扰动分析映射的GAL 11 P-结合位点的C-末端螺旋α 3和α 1和α 2之间的环。一个GAL 11 P单体结合到一个GAL 4-dd二聚体，使得二聚体不对称，并暗示极端的负协同机制。丙氨酸扫描诱变的GAL 4-dd表明，NMR衍生的GAL 11 P结合面是至关重要的新的转录激活功能的GAL 4-dd对GAL 11 P的相互作用。GAL 4与GAL 11 P的结合虽然是人工相互作用，但代表了能够结合单个靶标以影响基因表达的激活区的独特结构基序。
The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme. Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C-2 symmetry containing three helices that mediate dimerization via coiled-coil contacts. The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs. Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix alpha3 and the loop between alpha1 and alpha2. One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction. The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression.