Functional domains of human RAP74 including a masked polymerase binding domain

RAP74, the large subunit of human transcription factor IIF (TFIIF), has been analyzed by deletion mutagenesis and in vitro assays to map functional domains. Tight binding to the RAP30 subunit involves amino acids between positions 1-172. Amino acids 1-205 are minimally sufficient to stimulate accurate transcription from the adenovirus major late promoter in an extract system, although C-terminal sequences contribute to activity. A partially masked RNA polymerase II binding domain has been mapped to the C-terminal region of the protein (amino acids 363-444). Sequences near the N terminus and within the central portion of RAP74 affect accessibility of this domain. Extending this domain to 363-486 creates a peptide that binds polymerase and DNA and inhibits transcription initiation in vitro from non-promoter DNA sites. This larger C-terminal domain may modify polymerase interaction with template during initiation and/or elongation of RNA chains.