A Novel Zinc Finger Structure in the Large Subunit of Human General Transcription Factor TFIIE

Abstract

The zinc finger domain in the large subunit of TFIIE (TFIIE␣) is phylogenetically conserved and is essential for transcription. Here, we determined the solution structure of this domain by using NMR. It consisted of one ␣-helix and five ␤-strands, showing novel features distinct from previously determined zinc-binding structures. We created point mutants of TFIIE␣ in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Four Zn 2؉ -ligand mutants, in which each of cysteine residues at positions 129, 132, 154, and 157 was replaced by alanine, possessed no transcription activities on a linearized template, whereas, on a supercoiled template, interesting functional asymmetry was observed: although the C-terminal two mutants abolished transcription activity (<5%), the N-terminal two mutants retained about 20% activities. The N-terminal two mutants bound stronger to the small subunit of TFIIF than the wild type and the C-terminal two mutants were impaired in their binding abilities to the XPB subunits of TFIIH. These suggest that the structural integrity of the zinc finger domain is essential for the TFIIE function, particularly in the transition from the transcription initiation to elongation and the conformational tuning of this domain for appropriate positioning of TFIIF, TFIIH, and polymerase II would be needed depending on the situation and timing.In eukaryotes, transcription initiation of protein-coding genes requires RNA polymerase II (pol II) 1 and its auxiliary five general transcription factors, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (1, 2). Pol II and general transcription factors assemble together on a promoter DNA to form a preinitiation complex (PIC). TATA box-binding protein, a DNA binding subunit of TFIID, binds first to the TATA box. TFIIB then binds and recruits pol II to the complex (3). Pol II comes with TFIIF, which is important for accurate location of pol II in the complex around the transcription initiation site (4). Finally, TFIIE and TFIIH are incorporated to complete PIC formation (3).During PIC formation, TFIIE interacts with various general transcription factors, pol II, and promoter DNA, recruits TFIIH into the PIC, and regulates the enzymatic activities of TFIIH; serine kinase of the C-terminal domain of the largest subunit of pol II, DNA-dependent ATPase, and DNA helicase activities (5-7). At transcription initiation, TFIIE binds both to pol II near its active site and to promoter DNA ϳ10 bp upstream (Ϫ10) from the transcription initiation site (ϩ1), where the promoter melting starts (8). There TFIIE conducts pol II to sit at the initiation site, makes pol II processive upon C-terminal domain phosphorylation by stimulating C-terminal domain kinase activity of TFIIH, and simultaneously assists a helicase subunit XPB of TFIIH to start melting at the Ϫ10 position. At the transition from initiation to elongation, TFIIE also plays a direct role in promoter clearance by regulating kinase and DNA helicase activities o...

Near-atomic resolution visualization of human transcription promoter opening

“…TFIIEβ WH2 residues 150–207 were aligned with Sulfolobus tokodaii STO12A residues 25–106 (PDB: 2D1H) 73 . The human ZR domain, residues 125–164 (PDB: 1VD4) 74 , was docked near the base of the stalk domain of Pol II. The human TFIIEβ WH1 residues 75–146 (PDB: 1D8J) 75 were fitted into the region proximal to TFIIF RAP30 WH domain.…”

An Integrated Chemical Cross-linking and Mass Spectrometry Approach to Study Protein Complex Architecture and Function

“…The TFIIE small subunit Tfa2 contains two tandem winged helix domains (39,40). NMR structures of three conserved TFIIE domains have been determined (39,41,42), but only a low resolution EM structure of the TFIIE heterodimer is available (43). This is likely due to difficulties in obtaining diffraction quality crystals of the complete complex.…”

Structural basis of transcription initiation by RNA polymerase II

“…The structure of the archaeal homologue of the N-terminal part of TFIIEα, TFE, revealed an extended WH domain that had extra helices on the N and C termini, and indicated that the domain does not interact with DNA 133 . The essential zinc-finger domain from human TFIIEα has a novel topology 134 . The C-terminal acidic region forms a compact domain 135 that is required for strong interaction with TFIIH 131 , apparently through binding of the pleckstrin homology domain of p62 in TFIIH 135 .…”