A Key Role for the α1 Helix of Human RAP74 in the Initiation and Elongation of RNA Chains

Abstract

RNA polymerase II-associating protein 74 (RAP74) is the large subunit of transcription factor IIF (TFIIF), which is essential for accurate initiation and stimulates elongation by RNA polymerase II. Mutations within or adjacent to the alpha1 helix of the RAP74 subunit have been shown to decrease both initiation and elongation stimulation activities without strongly affecting the interactions of RAP74 with the RAP30 subunit or the interaction between TFIIF and RNA polymerase II. In this manuscript, mutations within the alpha1 helix are compared with mutations made throughout the neighboring conserved N-terminal domain of RAP74. Changes within the N-terminal domain include disruptions of specific contacts with the alpha1 helix, which were revealed in the recently published x-ray crystal structure (Gaiser, F., Tan, S., and Richmond, T. J. (2000) J. Mol. Biol. 302, 1119-1127). Contacts between the beta4-beta5 loop and the alpha1 helix are shown to be largely unimportant for alpha1 helix function. Other mutations throughout the N-terminal domain are consistent with the establishment of the dimer interface with the RAP30 subunit. The RAP74-RAP30 interface is important for TFIIF function, but no particular RAP74 amino acids within this region have been identified that are required for TFIIF activities. The molecular target of the alpha1 helix remains unknown, but our studies refocus attention on this important functional motif of TFIIF.