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Molecular and Cellular Biology, May 2005, p. 3583-3595, Vol. 25, No. 9
0270-7306/05/$08.00+0     DOI: 10.1128/MCB.25.9.3583-3595.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Human RNA Polymerase II Elongation in Slow Motion: Role of the TFIIF RAP74 {alpha}1 Helix in Nucleoside Triphosphate-Driven Translocation

Chunfen Zhang, Katie L. Zobeck,{dagger} and Zachary F. Burton*

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan

Received 1 December 2004/ Returned for modification 27 December 2004/ Accepted 31 December 2004

The role of the RAP74 {alpha}1 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 {alpha}1 helix, and RAP74(1-158), in which the {alpha}1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the {alpha}1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in {alpha}1. 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 {alpha}1 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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Michigan State University, 224 Biochemistry Building, East Lansing, MI 48824-1319. Phone: (517) 353-0859. Fax: (517) 353-9334. E-mail: burton{at}msu.edu.

{dagger} Present address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853-2703.

Molecular and Cellular Biology, May 2005, p. 3583-3595, Vol. 25, No. 9
0022-538X/05/$08.00+0     DOI: 10.1128/MCB.25.9.3583-3595.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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