RNA-POLYMERASE II-ASSOCIATED PROTEIN (RAP)-74 BINDS TRANSCRIPTION FACTOR (TF)-IIB AND BLOCKS TFIIB-RAP30 BINDING

A set of deletion mutants of human RNA polymerase II-associated protei n (RAP) 30, the small subunit of transcription factor IIF (TFIIF; RAP3 0/74), was constructed to map functional domains. Mutants were tested for accurate transcriptional activity, RAP74 binding, and TFIIB bindin g. Transcription assays indicate the importance of both N- and C-termi nal sequences for RAP30 function. RAP74 binds to the N-terminal region of RAP30 between amino acids 1 and 98. TFIIB binds to an overlapping region of RAP30, localized to amino acids 1-176 (amino acids 27-152 co mprise a minimal binding region), The C-terminal region of RAP74 (amin o acids 358-517) binds directly and independently to TFIIB, Interestin gly, RAP74 blocks TFIIB-RAP30 binding, both by binding TFIIB and by bi nding RAP30. When the TFIIF complex is intact, therefore, TFIIB TFIIF contact is maintained through RAP74. If the TFIIB-RAP30 interaction is physiologically important, the TFIIF complex must dissociate within s ome transcription complexes.