Gene expression profile of Tra1 GID (Gal4 interaction defective) mutants

Promoter-specific transcriptional activators (activators) stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the “target”. Previous studies have provided evidence that the Tra1 subunit of the yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is the target of the yeast activator Gal4. However, several other general transcription factors, in particular the mediator complex, have also been implicated as Gal4 targets. To investigate the essentiality of Tra1 as a target of Gal4, here we derive Tra1 mutants that are selectively defective for interaction with Gal4 in vivo (Gal4 Interaction Defective (GID) mutants). In contrast to wild-type Tra1, Tra1 GID mutants are not recruited by Gal4 to the promoter and cannot support Gal4-directed transcription activation, demonstrating that the Gal4–Tra1 interaction is required for Gal4 function. In yeast strains expressing a Tra1 GID mutant, Gal4 promoter binding is unexpectedly also diminished indicating that Gal4 and Tra1 bind cooperatively. Consistent with cooperative binding, we demonstrate that the interaction between Gal4 and Tra1 occurs predominantly on the promoter and not off DNA. Finally, we show that although Tra1 is also targeted by other activators, these interaction are unaffected by GID mutations, revealing an unanticipated specificity of the Gal4-Tra1 interaction. 3 samples were analyzed in duplicate with completely randomized design

启动子特异性转录激活因子（promoter-specific transcriptional activators，下称激活因子）通过与被定义为“靶标”的转录机器（transcription machinery）的一种或多种组分直接相互作用，激活转录过程。既往研究已证实，酵母SAGA（Spt-Ada-Gcn5-乙酰转移酶）复合物的Tra1亚基是酵母激活因子Gal4的作用靶标。不过，另有多种通用转录因子（general transcription factors）——尤其是中介体复合物（mediator complex）——也被证实可作为Gal4的靶标。为探究Tra1作为Gal4靶标的必要性，本研究构建了在体内与Gal4发生选择性相互作用缺陷的Tra1突变体（Gal4 Interaction Defective，简称GID突变体）。与野生型Tra1不同，Tra1 GID突变体无法被Gal4招募至启动子区域，也无法介导Gal4依赖的转录激活，这直接证明Gal4与Tra1的相互作用是Gal4行使转录激活功能所必需的。在表达Tra1 GID突变体的酵母菌株中，Gal4的启动子结合能力也出现了意料之外的减弱，这提示Gal4与Tra1之间存在协同结合效应。与协同结合的结论相符，我们证实Gal4与Tra1的相互作用主要发生在启动子区域，而非游离于DNA分子之外。最后，本研究发现尽管Tra1也可被其他激活因子靶向结合，但此类相互作用均不受GID突变的影响，这揭示了Gal4-Tra1相互作用此前未被认知的特异性。本研究采用完全随机化实验设计，对3份样本进行了双重复检测。