REGULATION OF THE HUMAN GENERAL TRANSCRIPTION INITIATION-FACTOR TFIIFBY PHOSPHORYLATION

The transcription initiation factor, TFIIF, is essential not only for the initiation of transcription but also for efficient elongation of m RNA synthesis by mammalian RNA polymerase II and is extensively phosph orylated in vivo. The possible regulation of TFIIF activity by protein phosphorylation was investigated by comparing the biochemical propert ies of alkaline phosphatase-treated HeLa TFIIF with those of native or bacterially expressed factor. Alkaline phosphatase treatment decrease d the size of the large subunit (RAP74) of TFIIF to that of the recomb inant protein but did not change the size of the small subunit (RAP30) . Both the transcription initiation and elongation stimulating activit ies of the alkaline phosphatase-treated TFIIF decreased to 15-20% of t he native form under conditions in which the amount of TFIIF was rate- limiting for transcription. Furthermore, phosphatase-treated TFIIF ass embled the DBPolF complex and bound to RNA polymerase Bless efficientl y than the native protein. When hybrid TFIIFs were reconstituted using native or recombinant subunits, a native form of RAP74 stimulated bot h transcription and DBPolF complex formation activity regardless of wh ether native or recombinant RAP30 was used. We propose that TFIIF acti vity is regulated by protein phosphorylation, particularly of the RAP7 4 subunit. The functional role of RAP74 in assembling the preinitiatio n complex and modulating TFIIF activity is discussed.