MECHANISM OF INITIATION AT RNA POLYMERASE II PROMOTERS

一套精确的基因表达程序控制着许多复杂的事物 生物过程，如细胞生长、分化和 发展。阐明正常对照的发病机制 在各种肿瘤、出生缺陷和其他疾病中流产， 因此需要对基因调控有一个基本的了解 表情。总的来说，RNA聚合酶II(PolII)的调节 基因的转录控制着它的表达。一组复杂的DNA POL启动转录所需的序列和蛋白质 II发起人。启动子序列可分为：1)和 起始区，从-35延伸到+20(其中+1是起始区 站点)，2)从-110延伸到-50的紧邻上游区域， 3)增强子序列，距离相差很大。这个 后两类序列的激活机制 由于它们的影响，抄写特别耐人寻味，也不清楚。 双向和可变距离。 这项提案的目标是定义直接上游的 PolII启动子区域可以刺激转录的启动。 对DNA和蛋白质成分的表征是有意的。 这些组件的活性将通过以下两方面的影响来衡量 在体内和体外转录，以及蛋白质-DNA相互作用， 部分通过快速硝化纤维滤膜结合试验进行测定。 建议项目的重点是SV40的早期和后期推动者。 这些启动子特别适合于此类研究，因为：1) 在体外和体内的转录强烈依赖于它们的共享 2)体外转录也很强 依赖于特定的细胞因子，该因子似乎通过 上游DNA序列，以及3)监测特定结合的检测 细胞因子对上游序列的影响已经被开发出来。 拟议项目的具体目标是1)确定SV40 直接上游序列可以刺激转录在不同的 相对于异源起始点的位置和取向，2) 分离和诱变SV40上游最小活性序列 区域，3)纯化特异性结合的细胞蛋白(S) SV40上游序列，4)确定是否有DNA结合蛋白(S) 参与转录，5)表征相互作用 脱氧核糖核酸结合蛋白(S)和SV40DNA，以及6)识别其他病毒 和/或与蛋白质相互作用的细胞启动子(S)。

A set of precise programs of gene expression control many complex biological processes, such as cell growth, differentiation, and development. An elucidation of the mechanisms by which the normal controls are aborted, as in various neoplasms, birth defects, and other diseases, will therefore require a basic understanding of the regulation of gene expression. In general, regulation of RNA polymerase II (pol II) transcription of a gene controls its expression. A complex set of DNA sequences and proteins are required for initiation of transcription at pol II promoters. The promoter sequences can be divided into: 1) an initiation region, extending from -35 to +20 (where +1 is the initiation site), 2) an immediate upstream region, extending from about -110 to -50, and 3) enhancer sequences, positioned at widely varying distances. The mechanisms by which the latter two classes of sequences activate transcription are particularly intriguing and unclear, due to their effects bidirectionally and at varying distance. The objective of this proposal is to define how the immediate upstream region of pol II promoters can stimulate the initiation of transcription. The characterization of both DNA and protein components is intended. Activity of these components will be measured by effects both on transcription, in vivo and in vitro, and on protein-DNA interactions, assayed in part by a rapid nitrocellulose filter binding assay. The focus of the proposed project is the SV40 early and late promoters. These promoters are uniquely suited to such studies because: 1) transcription in vitro and in vivo is strongly dependent on their shared immediate upstream region, 2) transcription in vitro is also strongly dependent on a specific cellular factor, which seems to function through the upstream DNA sequences, and 3) an assay which monitors specific binding of a cellular factor to the upstream sequences has been developed. The specific aims of the proposed project are 1) to determine if the SV40 immediate upstream sequences can stimulate transcription in varying positions and orientations relative to a heterologous initiation site, 2) to isolate and mutagenize the minimal active sequences of the SV40 upstream region, 3) to purify the cellular protein(s) which specifically binds the SV40 upstream sequences, 4) to determine whether the DNA-binding protein(s) is involved in transcription, 5) to characterize the interactions between the DNA-binding protein(s) and SV40 DNA, and 6) to identify other viral and/or cellular promoters which interact with the protein(s).