Mechanism of Termination In Vitro by RNA Polymerase II

该项目将在体外系统生化表征人类RNA聚合酶II在腺病毒主要晚期转录单元内的一个位点上的转录衰减机制。在腺病毒感染过程中，这个位点在后期而不是早期促进转录阻断。实验条件已经发现，可以在可溶性的无细胞蛋白提取物中观察和研究该位点的有效终止。本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。这一分析将补充和扩展的定位和解剖的DNA序列，有助于延伸行为的聚合酶。此外，一些功能性质的反应将被检查，以提供一个更好的理解终止的分子机制。这些特性包括转录物释放的动力学和程度，本研究的作用与最近来自许多不同实验室的证据有关，这些证据表明基因内的转录衰减是真核细胞中差异基因表达的重要调节机制。​本研究的长期目标是确定RNA聚合酶II在体内调节转录衰减的可能方法，以便未来的实验可以专门关注这些调节机制。* * * / /

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.*** //