Mechanism of Mediator Recruitment by Tandem Gcn4 Activation Domains and Three Gal11 Activator-Binding Domains
Mechanism of Mediator Recruitment by Tandem Gcn4 Activation Domains and Three Gal11 Activator-Binding Domains
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DOI：
10.1128/mcb.01046-09
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发表时间：
2010-05-15
影响因子：
5.3
通讯作者：
Hahn, Steven
中科院分区：
文献类型：
--
作者：
Herbig, Eric;Warfield, Linda;Hahn, Steven
通过定点交联法确定了转录预起始复合体中串联的GCN4酸性激活域的靶点。单独的Gcn4激活结构域与三个共同的靶标Gal11/MED15、Taf12和Tra1交链，这三个靶标是四个保守的共激活复合体Mediator、SAGA、TFIID和NuA4的亚单位。Gcn4 N端激活结构域也与介体亚单位Sin4/Med16交叉连接。这两个GCN4激活结构域对转录的贡献是基因特异性的，从协同作用到次要的相加作用。Gcn4依赖的基因对Gal11的需求从10倍依赖到完全Gal11独立，而Gcn4和Taf12的相互作用对所研究的任何基因的表达都没有显著贡献。互补方法确定了三个保守的Gal11激活子结合域，它们以微摩尔亲和力结合每个GCN4激活域。这些Gal11激活剂结合域对依赖Gcn4和Gal11的基因的转录激活和介体募集起到了额外的作用。虽然我们发现保守的Gal11 KIX结构域参与了Gal11的功能，但我们没有发现Gcn4-KIX特异性相互作用的证据，得出Gal11 KIX结构域不是通过与Gcn4特异性相互作用发挥作用的结论。我们的综合结果显示了基因特定的辅激活子需求，激活子-靶相互作用中令人惊讶的冗余，以及由多个低亲和力蛋白质-蛋白质相互作用介导的激活子-辅激活子相互作用。
Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activatorbinding domains contribute additively to transcription activation and Mediator recruitment at Gcn4-and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.