THE ADENOASSOCIATED VIRUS REP78 MAJOR REGULATORY PROTEIN BINDS THE CELLULAR TATA-BINDING PROTEIN IN-VITRO AND IN-VIVO

Rep78 is the major regulatory protein of adenoassociated virus (AAV). Rep78 is able to transcriptionally regulate all three of AAV's promote rs, as well as a variety of heterologous promoters. in an attempt to u nderstand the mechanism of action by which Rep78 is able to regulate g ene expression, we are investigating Rep78's possible protein-protein interaction with basal transcription factors. One such critical basal transcription factor is the human TATA binding protein, TBP. TBP is a core factor required for the assemblage of the transcription initiatio n complex, TFIID. In this report an in vitro interaction between Rep78 and TBP was demonstrated in three different assay systems, including West(far)-Western analysis, electrophoretic mobility shift assay-super shift, and coimmunoprecipitation. Furthermore, using the yeast GAL4 tw o-hybrid system, an in vivo interaction between Rep78 and TBP was also demonstrated. Further still. the amino half of Rep78 is shown to be n eeded for Rep78-TBP interaction. Mutations within this region of Rep78 are known to be defective for transcriptional regulatory ability, sug gesting a biological role for this interaction. Thus. Rep78 may regula te transcription through binding and regulating TBP's numerous interac tions. Furthermore, as Rep78 is known to bind at least one other trans cription factor (Sp1) and likely others, Rep78 may function as a TBP-a ssociated factor in an altered TFIID-like complex. (C) 1998 Academic P ress.