A novel zinc finger structure in the large subunit of human general transcription factor TFIIE.
The zinc finger domain in the large subunit of TFIIE (TFIIEalpha) is phylogenetically conserved and is essential for transcription. Here, we determined the solution structure of this domain by using NMR. It consisted of one alpha-helix and five beta-strands, showing novel features distinct from previously determined zinc-binding structures. We created point mutants of TFIIEalpha 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.
Okuda
Masahiko
M
Graduate School of Integrated Science, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Tanaka
Aki
A
Arai
Yoko
Y
Satoh
Manami
M
Okamura
Hideyasu
H
Nagadoi
Aritaka
A
Hanaoka
Fumio
F
Ohkuma
Yoshiaki
Y
Nishimura
Yoshifumi
Y
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019473:Transcription Factors, TFII / Q000737:chemistry*; D014158:Transcription, Genetic; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers
D017931:DNA Primers; D008024:Ligands; D011994:Recombinant Proteins; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D005982:Glutathione Transferase; D015032:Zinc; D003545:Cysteine; D000409:Alanine