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 ␣1 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 ␣1 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 ␣1 helix, which were revealed in the recently published x-ray crystal structure (Gaiser, F., Tan TFIIF1 appears to be an ␣␤ heterodimer of RAP74 and RAP30 subunits, and previous reports that TFIIF may be an ␣ 2 ␤ 2 heterotetramer are not supported by the x-ray crystal structure (1). Although not evident from primary sequence, RAP74 and RAP30 subunits are structurally similar, with an intricate series of N-terminal␤-sheets that form a RAP74-RAP30 dimer interface. RAP74 and RAP30 also have similar C-terminal regions with winged helix-turn-helix structures (2, 3). The larger size of the human RAP74 subunit can be attributed to an extensive loop rich in Gly, Pro, Ser, Thr, and charged residues separating more structured N-and C-terminal domains (4, 5).TFIIF is an RNA polymerase II-specific transcription factor restricted to the eukaryotic kingdom. Fig. 1, therefore, shows an amino acid sequence alignment of the N-terminal regions of several RAP74 homologues spaced throughout eukaryotic evolution. Beneath the alignment, the primary sequence is correlated with regions of secondary structure. Regions of ␣-helix and ␤-sheet are derived from the crystal structure of human TFIIF (1) or else from secondary structure predictions (6 -9) in the regions where structural information is not available. The x-ray crystal structure indicates a unique dimer interface made up of RAP74 ␤-sheets ␤1, ␤2, ␤3, ␤6, ␤7, ␤8, and the corresponding ␤-sheets in RAP30. The ␤4 and ␤5 sheets of RAP74 interact to form the structured base of a loop that diverges from the dimer core. In the crystal structure, the RAP74 ␣1 helix makes intimate contacts with the ␤4 and ␤5 sheets.The ␣1 helix has been shown previously to be highly sensitive to mutation (10, 11). Several single amino acid changes, particularly in hydrophobic residues, cause significant defects in both accurate initiation and elongation. Because of symmetrical effects on initiation and elongation, ␣1 should function by contacting a molecular target common to both processes, which would implicate RNA polymerase II, TFIIF, and DNA as the most likely targets. Mutations in ␣1 do not appear to affect interaction with the RAP30 subunit, and no RAP30 contacts are identified for ␣1 in the crystal structure (1). As far as can be discerned, ␣1 mutations do not have an effect on assembly of gel-shif...

Conserved architecture of the core RNA polymerase II initiation complex

“…NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5310 ARTICLE Published mutational and kinetic data revealed important roles of the charged helix in transcription initiation and elongation 25,26 . These roles can now be rationalized due to the location near downstream DNA (Fig.…”

“…The charged helix was partially resolved in the X-ray structure 16 and is predicted to be longer 34 . Residues required for normal transcription initiation and elongation 25 are indicated as grey asterisks.…”

Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

“…Based on transient state kinetic studies, our laboratory recently proposed a mechanism for RNAP II elongation stimulated by TFIIF and hepatitis ␦ antigen (HDAg) (5). We have also demonstrated the major defects of the RAP74 I176A mutant in general and transient state elongation assays, providing insight into the functions of TFIIF in the RNAP II mechanism (11). We find that TFIIF exerts a global effect on elongation.…”

“…Rapid Quench Elongation Experiments-Detailed protocols for rapid quench-flow experiments have been published previously (4,5,11). Briefly, 32 P-labeled C40 (40-nucleotide RNA ending in a 3Ј-CMP) RNAP II ECs were formed on metal bead-immobilized templates in a HeLa transcription extract.…”

“…TFIIF helps commit elongation complexes (ECs) to the forward synthesis pathway. TFIIF stimulates the rate of chemistry and accelerates a slow step after chemistry in the normal processive transition between bonds, a step that includes translocation and pyrophosphate release (5,11). We conjecture that TFIIF binds to an external surface of RNAP II, tightens the RNAP II clamp, which grasps the RNA-DNA hybrid, and optimizes conditions for chemistry during elongation.…”