EUKARYOTIC TRANSCRIPTION COMPLEXES

真核生物蛋白质编码基因的转录起始涉及含有RNA聚合酶II、SRB/介体组分、六种通用转录因子（GTF）和启动子DNA的多蛋白质-DNA复合物的组装。 完全组装的转录起始复合物含有超过50种不同的多肽，分子量超过3 MDa。拟议的工作解决了转录起始复合物的结构，组装的复合物的途径，和启动子序列元件，管理的稳定性，组装和功能的复合物。 具体目标1：利用系统的位点特异性蛋白质-DNA光交联来绘制转录起始复合物中蛋白质-DNA相互作用的图谱。 具体目标二：使用动力学位点特异性蛋白质-DNA光交联来确定转录起始复合物内蛋白质-DNA相互作用形成的动力学。 具体目标3：使用结合位点选择来定义GTF识别的启动子序列元件。 具体目标4：利用高密度阵列表达分析确定GTF识别的启动子序列元件的功能意义。这些结果将有助于理解人类的转录起始，理解人类的转录调控，并设计和合成低分子量的蛋白质-DNA相互作用的抑制剂在人类的转录起始，在体外和体内的转录起始的选择性调节中的应用，并可能在选择性的抗病毒和抗癌治疗中的应用。

Transcription initiation at a eukaryotic protein-encoding gene involves assembly of a multiprotein-DNA complex containing RNA polymerase II, SRB/Mediator components, six general transcription factors (GTFs), and promoter DNA. The fully assembled transcription initiation complex contains more than 50 distinct polypeptides and has a molecular mass of more than 3 MDa. The proposed work addresses the structure of the transcription initiation complex, the pathway of assembly of the complex, and the promoter sequence elements that govern stability, assembly, and function of the complex. The proposed work has four specific aims: Specific Aim 1: To use systematic site-specific protein-DNA photocrosslinking to map protein-DNA interactions within the transcription initiation complex. Specific Aim 2: To use kinetic site-specific protein-DNA photocrosslinking to define the kinetics of formation of protein-DNA interactions within the transcription initiation complex. Specific Aim 3: To use binding-site selection to define promoter sequence elements recognized by GTFs. Specific Aim 4: To use high-density-array expression analysis to define the functional significance of promoter sequence elements recognized by GTFs. The results to be obtained will contribute to understanding human transcription initiation, to understanding human transcription regulation, and to design and synthesis of low-molecular-weight inhibitors of protein-DNA interactions in human transcription initiation, for application in selective regulation of transcription initiation in vitro and in vivo, and possibly for application in selective antiviral and anticancer therapy.