Mechanism of Termination In Vitro by RNA Polymerase II

这项拟议的项目将是对腺病毒主要晚期转录单位内一个位置的人RNA聚合酶II转录减弱机制的体外系统生化表征。在腺病毒感染过程中，该位点已被证明在晚期但不是早期促进转录阻断。已经找到了在可溶性无细胞蛋白质提取物中观察和研究在该位置有效终止的实验条件。这项研究的一个主要目标是确定参与该位点终止或反终止的蛋白质，并表征这些蛋白质在反应机制中的作用。这一分析将通过定位和解剖DNA序列来补充和扩展，这些DNA序列有助于聚合酶的延伸行为。此外，还将研究该反应的一些功能性质，以更好地了解终止的分子机制。这些特性包括转录释放的动力学和程度，这项研究的作用与来自许多不同实验室的最新证据有关，这些证据表明，基因内的转录衰减是真核细胞差异基因表达的重要调节机制。这项研究的长期目标是确定在体内可能调节RNA聚合酶II抑制转录的可能方式，以便未来的实验可以专门专注于确定这些调节机制。

The proposed project will be a systematic biochemical characterization in vitro of the mechanism of transcription attenuation by human RNA polymerase II at a site within the adenovirus major late transcription unit. This site has been shown to promote transcriptional blockage at late but not early times during adenovirus infection. Experimental conditions have been found with which efficient termination at this site can be observed and studied in a soluble, cell-free protein extract. A major goal of this research is to identify the proteins that are involved in termination or antitermination at this site and to characterize the role of these proteins in the reaction mechanism. This analysis will be complemented and extended by a localization and dissection of the DNA sequences that contribute to the elongation behavior of the polymerase. In addition, a number of functional properties of the reaction will be examined to provide a better understanding of the molecular mechanism of termination. These properties include the kinetics and extent of transcript release and the role of this research relates to recent evidence from a number of different laboratories that transcription attenuation within genes is an important regulatory mechanism in differential gene expression in eukaryotic cells. The long-term goal of this research is to identify possible ways attenuation of transcription by RNA polymerase II might be regulated in vivo so that future experiments can focus specifically on defining these regulatory mechanisms.*** //