FUNCTIONS OF THE N-TERMINAL AND C-TERMINAL DOMAINS OF HUMAN RAP74 IN TRANSCRIPTIONAL INITIATION, ELONGATION, AND RECYCLING OF RNA-POLYMERASE-II

Transcription factor IIF (TFIIF) cooperates with RNA polymerase II (po l II) during multiple stages of the transcription cycle including prei nitiation complex assembly, initiation, elongation, and possibly termi nation and recycling. Human TFIIF appears to be an alpha(2) beta(2), h eterotetramer of RNA polymerase II-associating protein 74- and 30-kDa subunits (RAP74 and RAP30). From inspection of its 517-amino-acid (aa) sequence, the RAP74 subunit appears to comprise separate N- and C-ter minal domains connected by a flexible loop. In this study, we present functional data that strongly support this model for RAP74 architectur e and further show that the N- and C-terminal domains and the central loop of RAP74 have distinct roles during separate pleases of the trans cription cycle. The N-terminal domain of RAP74 (minimally aa 1 to 172) is sufficient to deliver pal II[ into a complex formed on the adenovi rus major late promoter with the TATA-binding protein, TFIIB, and RAP3 0, A more complete N-terminal domain fragment (aa 1 to 217) strongly s timulates both accurate initiation and elongation by pal II, The regio n of RAP74 between aa 172 and 205 and a subregion between aa 170 and 1 78 are critical for both accurate initiation and elongation, and mutat ions in these regions have similar effects on initiation and elongatio n. Based on these observations, RAP74 appears to have similar function s in initiation and elongation, The central region and the C-terminal domain of RAP74 do not contribute strongly to single-round accurate in itiation or elongation stimulation hat do stimulate multiple-round tra nscription in an extract system.