Analysis of the mechanism of eukaryotic transcription initiation and its regulation based on the TATA box-binding factor TFIID

TATA盒结合因子TFIID在转录激活机制中起着至关重要的作用。我们通过多种方法阐明了TFIID的功能和结构在真核生物转录起始及其调控中的意义。1)编码人TFIID亚基的cDNA的分离与鉴定分离到了两种编码人TFIID亚基的新型cDNA，它们与组蛋白具有相似性。分析了这些亚基与其他TFIID亚基之间的相互作用。我们利用与组蛋白H4相似的其他TFIID亚基作为探针，分离出与组蛋白H2B结构相似的TFIID亚基，发现两个亚基的相互作用是由它们各自的组蛋白折叠基序引起的。我们还分离了其他类似于组蛋白H2A的TFIID亚基，并展示了组蛋白10月样结构模型。3)从多个物种中分离编码TFIID亚基的cDNA为了阐明其结构与功能之间的关系，我们从多个物种中分离了编码TFIID亚基的cDNA。4) S.pombe TFIID亚基的遗传分析为了阐明TFIID亚基在体内的功能，我们分离了S.pombe TFIID亚基。提出了一种分离温度敏感突变体的新方法。分离的突变体TFIID亚基在胞质分裂中表现出缺陷。5)分离编码与TFIID亚基相互作用的各种新因子的cdna分离编码与TFIID亚基相互作用的各种新因子的cdna。这些分离的因子大多与染色质有关。其中一些具有组蛋白乙酰转移酶活性和核小体重塑活性。

TATA box-binding factor TFIID plays a crucial role in machanisms of transcription activation.We elucidated the significance of function and structure of TFIID in eukaryotic transcription initiation and its regulation by several methods.1)Isolation and characterization of cDNA encoding human TFIID subunitsTwo novel types of cDNAs, which encode human TFIID subunits and have similalities to histones, were isolated. The analysis of interactions between these subunits and other TFIID subunits has been made.2)Model of histone octamer-like structure within TFIIDWe isolated TFIID subunit whose structure is similar to that of histone H2B using other TFIID subunit with histone H4 similarity as a probe and showed that the interaction of two subunits is caused by their own histone folding motifs. We also isolated other TFIID subunit similar to histone H2A and showed a model of histone octomer-like structure.3)Isolation of cDNA encoding TFIID subunits from several speciesWe isolated cDNA encoding TFIID subunits from several species in order to elucidate the relationship between structure and function.4)Genetical analysis of S.pombe TFIID subunitsTo elucidate the in vivo function of TFIID subunits, we isolated S.pombe TFIID subunits. New method for isolation of temparature sensitive mutants are developed. Isolated ts mutant TFIID subunit shows defect in cytokinesis.5)Isolation of cDNAs encoding various new factors interacting with TFIID subunitscDNAs encoding various new factors interacting with TFIID subunits were isolated. Most of these isolated factors are chromatin-related. Some of them are shown to have histone acetyl transferase activities and nucleosome-remodeling activities.