EUKARYOTIC TRANSCRIPTION COMPLEXES

真核蛋白编码基因的转录起始涉及多蛋白-DNA复合体的组装，该复合体包含RNA聚合酶II、SRB/Mediator组分、6个通用转录因子（GTFs）和启动子DNA。完全组装的转录起始复合物包含50多个不同的多肽，分子量超过3mda。提出的工作涉及转录起始复合物的结构，复合物的组装途径，以及控制复合物稳定性，组装和功能的启动子序列元件。提出的工作有四个具体目标：具体目标1：使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内蛋白质- dna相互作用。特定目标2：使用动态位点特异性蛋白质- dna光交联来定义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。研究结果将有助于理解人类转录起始，理解人类转录调控，设计和合成人类转录起始蛋白- dna相互作用的低分子量抑制剂，应用于体外和体内转录起始的选择性调控，并可能应用于选择性抗病毒和抗癌治疗。

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.