Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate-driven translocation.
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.
Zhang
Chunfen
C
Department of Biochemistry and Molecular Biology, Michigan State University, 224 Biochemistry Building, East Lansing, MI 48824-1319, USA.
Zobeck
Katie L
KL
Burton
Zachary F
ZF
D002478:Cells, Cultured; D006801:Humans; D009711:Nucleotides / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D012319:RNA Polymerase II / Q000502:physiology*; D017384:Sequence Deletion / Q000235:genetics; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics / Q000502:physiology*
D009711:Nucleotides; D019473:Transcription Factors, TFII; D012319:RNA Polymerase II; C056604:transcription factor TFIIF