Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II

The general transcription factor IIF (TFIIF) assembled in the initiation co mplex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsib le for the interaction. We examined whether TFIIF interacts with RNA polyme rase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulat ory protein, RPB5-mediating protein. The results demonstrated that RPB5 dir ectly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30- binding region was mapped to the central region (amino acids (aa) 47-120) o f RPB5, which partly overlaps the hepatitis B virus X protein-binding regio n. Although the middle part (aa 101-170) and the N-terninus (aa 1-100) of R AP30 independently bound RPB5, the latter was not involved in the RPB5 bind ing when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substituti ons pinpointed two residues critical for the RPB5 binding in in vitro and i n vivo assays. Wild type hut not mutants Y124A and Q131A of RAP30 coexpress ed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74- bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the cen tral region of RPB5 and wild type RAP30 inhibited recovery of endogenous po l II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-b inding region of RPB5 inhibited the association of TFIIF and pol II. The ex posed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.