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.*** //
