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 生物过程，如细胞生长、分化和 发展 阐明了正常对照的机制 流产，如各种肿瘤，出生缺陷和其他疾病， 因此，需要对基因的调控有基本的了解， 表情 一般来说，RNA聚合酶II（pol II） 基因的转录控制其表达。 一组复杂的DNA 转录起始需要序列和蛋白质， II启动子。 启动子序列可以分为：1）启动子序列， 起始区，从-35延伸到+20（其中+1是起始区 位点），2）紧邻上游区域，从约-110延伸至-50， 和3）增强子序列，位于广泛变化的距离处。 的 后两类序列激活的机制 转录特别有趣和不清楚，由于它们的影响， 双向地且在不同的距离处。 本提案的目的是确定直接上游 pol II启动子的区域可以刺激转录起始。 预期对DNA和蛋白质组分进行表征。 这些成分的活性将通过对 转录，在体内和体外，和蛋白质-DNA相互作用， 部分通过快速硝酸纤维素滤膜结合试验测定。 拟议项目的重点是SV 40早期和晚期启动子。 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 直接上游区域，2）体外转录也强烈 依赖于一种特定的细胞因子，它似乎通过 上游DNA序列，和3）监测特异性结合的测定 细胞因子与上游序列的相互作用。 拟议项目的具体目标是：1）确定SV 40是否 直接上游序列可以刺激转录， 相对于异源起始位点的位置和方向，2） 分离并诱变SV 40上游的最小活性序列 3）纯化特异性结合所述区域的细胞蛋白， SV 40上游序列，4）以确定DNA结合蛋白是否 参与转录，5）表征之间的相互作用 DNA结合蛋白和SV 40 DNA，以及6）鉴定其它病毒 和/或与蛋白质相互作用的细胞启动子。

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).