Journal of Biological Chemistry
Volume 279, Issue 49, 3 December 2004, Pages 51395-51403
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Protein Structure and Folding
A Novel Zinc Finger Structure in the Large Subunit of Human General Transcription Factor TFIIE*

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The zinc finger domain in the large subunit of TFIIE (TFIIEα) is phylogenetically conserved and is essential for transcription. Here, we determined the solution structure of this domain by using NMR. It consisted of one α-helix and five β-strands, showing novel features distinct from previously determined zinc-binding structures. We created point mutants of TFIIEα in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Four Zn2+-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.

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The atomic coordinates and structure factors (code 1VD4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported by a Collaborative of Regional Entities for the Advancement of Technological Excellence (CREATE) from Japan Science and Technology agency, by a Project of Protein 3000, Transcription and Translation, and by Grants in Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table S1: Coupling constants for 3JHNHα, Fig. S1: Sequential and medium range NOEs, Fig. S2: Chemical shift indices, Fig. S3: Beta sheet diagram with long range (dαα, dαN and dNN) NOEs, Fig. S4: 2D CPD-H(N)CO spectra for the detection of trans-hydrogen bond 3hJNC′ couplings, and Fig. S5: Ramachandran diagram.

**

Present address: Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.

‡‡

Present address: RIKEN, Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.