Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate-driven translocation.

ABSTRACT

The role of the RAP74 alpha1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1-227), which includes an intact alpha1 helix, and RAP74(1-158), in which the alpha1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the alpha1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in alpha1. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF 74(1-227) + TFIIS and TFIIF 74(1-158) + TFIIS. TFIIF 74(1-158) is defective because it fails to promote forward translocation. Deletion of the RAP74 alpha1 helix results in increased occupancy of the backtracking, cleavage, and restart pathways at a stall position, indicating reverse translocation of the elongation complex. During elongation, TFIIF 74(1-158) fails to support detectable nucleoside triphosphate (NTP)-driven translocation from a stall position and is notably defective in supporting bond completion (NTP-driven translocation coupled to pyrophosphate release) during the processive transition between bonds.