TFIIH HELICASES AND RNA POLYMERASE II HOLOENZYME

转录调控是生物学和细胞的基础。 健康生理学和疾病生理学。在拟议的项目中， 将研究转录反应的生物学机制。我们的 在基本转录的特征方面表现出专业知识 这些因素将是这些研究成功的关键。三大 将对问题进行调查。首先，解旋酶活性是一种基本的 在启动基础转录时的要求将是 研究其在基础转录和激活转录中的作用以及在 伸长率。TFIIH亚基的解旋酶结构域将被修饰， 将其导入细胞，回收用于生化分析。第二, 新发现的哺乳动物RNA聚合酶H全酶将是 学习。基于酵母细胞中揭示的范例，全酶 可能负责大部分(如果不是全部)信使核糖核酸的产生。使用 疱疹病毒基因激活剂，VP16，作为一个强大的模型，我们将 用来描述转录激活的因子要求 全酶。我们还将决定全酶的命运 转录反应中的亚基。第三，具体 核因子-kappaB的转录激活将使用我们的 高纯度的系统。核因子-kappaB参与了活化 广泛的启动子对不同的环境线索作出反应 以及成熟B细胞中抗体的结构性产生。核因子- KappaB与rel癌基因家族有同源性，并在 影响转录水平的相互作用网络。直接推动者 核因子-kappaB的激活将在体外概括，该因子 与强大的模型激活剂GAL4相比- VP16。这些实验的结果将有助于阐明生物化学 基因表达调控的基础。

The regulation of transcription is fundamental to biology and cell physiology in health and in disease. In the proposed project, the biological mechanisms of the transcription reaction will be studied. Our demonstrated expertise in the characterization of the basal transcription factors will be a key to the success of these studies. Three major questions will be investigated. First, helicase activity, a fundamental requirement in the initiation of basal transcription, will be investigated for its role in basal and activated transcription and in elongation. The helicase domains of TFIIH subunits will be modified, transfected into cells, and recovered for biochemical analysis. Second, the newly identified mammalian RNA polymerase H holoenzyme will be studied. Based upon the paradigm revealed in yeast cells, the holoenzyme may be responsible for most, if not all, of mRNA production. Using the herpes viral gene activator, VP16, as a powerful model, we will characterize the factor requirements for activation of transcription with the holoenzyme. We will also determine the fate of the holoenzyme subunits during the transcription reaction. Third, the specific activation of transcription by NF-kappaB will be investigated using our highly purified system. NF-kappaB has been implicated in the activation of a wide range of promoters in response to diverse environmental cues and in the constitutive production of antibodies in mature B cells. NF- kappaB has homology to the rel oncogene family and plays a role in a network of interactions that affect transcription levels. Direct promoter activation by NF-kappaB will be recapitulated in vitro and the factor requirements compared to those of the powerful model activator, GAL4- VP16. Results from these experiments will help clarify the biochemical basis of the regulation of gene expression.