Yuan C X, Gurley W B
Department of Microbiology and Cell Science, Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville 32611-0700, USA.
Cell Stress Chaperones. 2000 Jul;5(3):229-42. doi: 10.1379/1466-1268(2000)005<0229:ptfhwt>2.0.co;2.
Protein-protein interactions between human heat shock transcription factor 1 (hHSF1) and general transcription factors TFIIA-gamma, TFIIB, TBP, TAF(II)32, and TAF(II)55 and positive coactivator PC4 were characterized in order to identify potential targets of contact in the transcriptional preinitiation complex. These contacts represent one of the final steps in the signal transfer of heat stress to the transcriptional apparatus. TATA-binding protein (TBP) and transcription factor IIB (TFIIB) were identified as major targets for HSF1 transcriptional activation domains AD1 and AD2 based on in vitro interaction assays. TBP showed affinity for AD2 and a fragment containing AD1, while the core domain of TFIIB interacted primarily with the AD1 fragment. Interactions were also detected between full-length HSF1 and the small subunit (gamma) of TFIIA. PC4 interacted weakly with HSF2 and showed even less affinity for HSF1. Coimmunoprecipitation of transiently expressed TBP in HeLa cells demonstrated that HSF1 AD2 and AD1+AD2 are able to bind TBP in vivo. Assays based on transcriptional interference confirmed predictions that both TBP and TFIIB can interact with HSF1 activation domains in HeLa cells. The negative regulatory region (NR) of HSF1 did not interact with any general factors tested in vitro but did bind TFIID in nuclear extracts through contacts that probably involve TATA associated proteins (TAFs). These results suggest a model for transcriptional regulation by HSF1 that involves a shift between formation of dysfunctional TFIID complexes with the NR and transcriptionally competent complexes with the C-terminal activation domains.
为了确定转录起始前复合物中潜在的接触靶点，对人类热休克转录因子1（hHSF1）与通用转录因子TFIIA-γ、TFIIB、TBP、TAF（II）32和TAF（II）55以及正向共激活因子PC4之间的蛋白质-蛋白质相互作用进行了表征。这些接触代表了热应激信号传递到转录装置的最后步骤之一。基于体外相互作用试验，TATA结合蛋白（TBP）和转录因子IIB（TFIIB）被确定为HSF1转录激活域AD1和AD2的主要靶点。TBP对AD2和包含AD1的片段表现出亲和力，而TFIIB的核心结构域主要与AD1片段相互作用。在全长HSF1与TFIIA的小亚基（γ）之间也检测到了相互作用。PC4与HSF2的相互作用较弱，对HSF1的亲和力更低。HeLa细胞中瞬时表达的TBP的共免疫沉淀表明，HSF1 AD2和AD1+AD2能够在体内结合TBP。基于转录干扰的试验证实了TBP和TFIIB都能在HeLa细胞中与HSF1激活域相互作用的预测。HSF1的负调控区（NR）在体外不与任何测试的通用因子相互作用，但通过可能涉及TATA相关蛋白（TAFs）的接触在核提取物中与TFIID结合。这些结果提出了一种HSF1转录调控模型，该模型涉及与NR形成功能失调的TFIID复合物和与C端激活域形成转录活性复合物之间的转变。
