CHARACTERIZATION OF THE INTERACTION BETWEEN THE ACIDIC ACTIVATION DOMAIN OF VP16 AND THE RNA-POLYMERASE-II INITIATION-FACTOR TFIIB

Contact between a transcriptional activator and one or more components of the RNA polymerase II transcription Initiation machinery is genera lly believed important for activators to function. Several different m olecular targets have been suggested for direct contact by herpes simp lex virus virion protein VP16, including the general initiation factor TFIIB. in this report we have used several strategies to critically a ssess this interaction between VP16 and TFIIB. Affinity columns of VP1 6 hound TFIIB activity from HeLa cell extracts and the binding was red uced by mutations in the activation domain of VP16, In assays of direc t binding, VP16 bound recombinant human TFIIB but not Drosophila or ye ast TFIIB, Unlike binding from an extract, however we found that the i nteraction between VP16 and recombinant human TFIIB was not affected b y mutations in VP16 that reduce transactivation, Point mutations withi n human TFIIB that reduce transactivation by VP16 have been shown to r educe VP16 binding, but we show here that these same mutations critica lly affect both the important TBP-TFIIB interaction and the ability of TFIIB to support activator-independent basal transcription in vitro, Taken together our results suggest more evidence is needed to support the notion that TFIIB is a functionally important target for the activ ator VP16.