9419311 Dynan In eukaryotic cells, the synthesis of a gene-specific messenger RNA is accomplished by transcription of the corresponding DNA by the enzyme RNA Polymerase II. Additional proteins called transcription factors are required to initiate and regulate transcription. The human retrovirus protein Tax increases the rate at which RNA Polymerase II transcribes certain genes to make messenger RNAs. Tax works by an unusual mechanism in that it increases the ability of a variety of transcription factors to interact with DNA This research will explore in detail the mechanism by which Tax enhances the interaction of various transcription factors with DNA, and the mechanism by which Tax activates transcription of the human retrovirus HTLV-I. The results of this research should greatly increase our understanding of the functional and regulatory interactions of eukaryotic transcription factors. *** The transcriptional regulatory protein, Tax, which is encoded by a human retro virus, increases the rate retrovirus at which RNA polymerase II transcribes certain genes, and does so by an unusual mechanism. Purified Tax protein increases the ability of other transcription factors to bind to DNA. Tax enhances the binding of the cyclic AMP response element binding protein (CREB), related proteins in the CREB/ATF family, and a number of factors that belong to other, unrelated, structural classes. In some cases, Tax enters into stable ternary complexes with other transcription factors and DNA but in other cases, ternary complexes have not been detected. In this research, the mechanism by which Tax enhances transcription factor binding will be explored in more detail. A new type of binding assay will be developed, which will allow direct, quantitative measurements of the association of Tax into ternary complexes with CREB and DNA. The assay will be used to study the effect of mutations in CREB and DNA on ternary complex formation. In separate experiments, a novel, highly specific UV crosslinking method w ill be used to characterize changes in CREB-DNA contacts in the presence of Tax. Experiments will also be performed to test a model where Tax preferentially interacts with the unfolded CREB DNA binding domain in order to guide the protein to DNA. It is important to establish which aspects of the Tax-CREB interaction are specific to CREB and which are generah To address this, the interaction of Tax with alternative target proteins, outside the CREB/ATF family, will be studied. Quantitative studies will be performed to characterize the effect of Tax on the DNA binding activity of Fos and Jun, Spl, and Etsl, Protein-protein interactions between Tax and the alternative target proteins will be characterized, and the model that Tax preferentially associates with unfolded proteins will again be tested. Finally, the mechanism by which Tax activates HTLV-I transcription will be investigated. It will be determined whether Tax-CREB complexes recruit additional transcription factors to the template DNA. We will perform preliminary investigations of the mechanism by which a small-molecule inhibitor blocks HTLV-I transcription. %%%
