9723391 Vales The role of transcriptional activation in regulating gene expression in eukaryotes has been amply documented. In contrast, the regulatory role of transcriptional repression in eukaryotes has been appreciated only lately based on the identification and role of several factors that function to inhibit transcription. Many important mechanistic studies in prokaryotes have revealed that repressors evolved to target almost every possible step in the transcription process. In contrast, given the new appreciation for repressors in the eukaryotic field, the mode of action of these factors is largely unknown. The focus of this research is the cellular protein RBP, which was originally isolated as a presumed recombinase. Studies of viral gene expression led to the identification of RBP as a transcriptional repressor. Recent studies have demonstrated that RBP targets the adenoviral gene encoding polypeptide IX (pIX). The newly recognized role of RBP as a transcriptional repressor presents an unique opportunity to identify the target(s) of RBP in repression as well as the mechanism by which repression is achieved. The viral pIX promoter is relatively simple, containing a single recognition site for the cellular transcription factor SP1. RBP binds immediately upstream of the TATA box in the viral promoter but does not dislodge the adjacent factor; therefore repression does not involve occlusion of adjacent factor binding. On the other hand, repression is dependent upon both the presence and the position of the RBP site in the pIX promoter. The goal of this research is to identify the precise target(s) of RBP in repression and to determine the molecular mechanism by which RBP mediates repression of the pIX gene. Experiments performed in vitro will identify the target(s) of RBP repression and will test if RBP cobinding with TFIID occludes binding by a third factor, TFIIA, required for activation. Possible interaction between RBP and SP1 will be examined using GST-pull down and coimm unoprecipitation experiments. If RBP interacts with SP1, the SP1 and RBP protein domains involved in this interaction will be identified. Possible interaction between RBP and general transcription factors required for activation will be probed for in a similar manner. Finally, the molecular mechanism by which RBP functions to repress transcription will be identified in a reconstituted transcription assay. The results from these studies will lay the groundwork for studying the means by which repression is relieved to allow optimal activation of viral pIX gene expression. %%% The role of transcriptional activation in regulating gene expression in eukaryotes has been amply documented. In contrast, the regulatory role of transcriptional repression in eukaryotes has been appreciated only lately based on the identification and role of several factors that function to inhibit transcription. The focus of this research is the cellular protein RBP, which was originally isolated as a presumed recombinase. The actual functional role of RBP as transcription factor was uncovered within the last three years. This research will exploit the viral pIX gene to understand the means by which RBP represses transcription in mammalian cells. The results from these studies will lay the groundwork for studying the means by which repression is relieved to allow optimal activation of viral pIX gene expression. ***
