ROLES FAR BOTH THE RAP30 AND RAP74 SUBUNITS OF TRANSCRIPTION FACTOR IIF IN TRANSCRIPTION INITIATION AND ELONGATION BY RNA-POLYMERASE-II

Mammalian transcription factor IIF (TFIIF) is a heterodimer composed o f similar to 30-kDa (RAP30) and similar to 70-kDa (RAP74) subunits. TF IIF has been shown to bind RNA polymerase II and control the activity of the enzyme in both the initiation and elongation stages of transcri ption. Although previous studies have established a role for RAP30 in assembly of the preinitiation complex and in transcription initiation, information on the function of RAP74 in these processes has been lack ing. Using a highly purified transcription system and assays that perm it sensitive measurement of the contributions of both RAP30 and RAP74 to TFIIF function, we have investigated the roles of these TFIIF subun its in transcription initiation and elongation. Results of template co mpetition experiments indicate that both RAP30 and RAP74 contribute to the formation of stable preinitiation intermediates containing RNA po lymerase II. Investigation of the role of TFIIF in transcription initi ation indicates that both RAP30 and RAP74 function in synthesis of the first few phosphodiester bonds of nascent transcripts and in formatio n of Sarkosyl-resistant preinitiation intermediates. Finally, kinetic experiments indicate that both RAP30 and RAP74 function in TFIIF-media ted stimulation of the rate of RNA chain elongation by RNA polymerase II.