© 2005 The Japanese Biochemical Society
Regular Paper |
Structural Insights into the Asymmetric Effects of Zinc-Ligand Cysteine Mutations in the Novel Zinc Ribbon Domain of Human TFIIE
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1 International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045; 2 Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045; 3 Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871; 4 Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871; and 5 Cellular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198
To whom correspondence should be addressed. Tel: +81-45-508-7216, Fax: +81-45-508-7362, E-mail: nisimura{at}tsurumi.yokohama-cu.ac.jp
The large subunit of TFIIE (TFIIE
) has a highly conserved zinc ribbon domain, which is essential for transcription. Recently, we determined the solution structure of this domain to be that of a novel zinc finger motif [Okuda et al. (2004) J. Biol. Chem. 279, 51395–51403]. On examination of the functions of four cysteine mutants of TFIIE, in which each of four zinc-liganded cysteines was replaced by alanine, we found an interesting functional asymmetry; on a supercoiled template, the two C-terminal mutants did not show any transcriptional activity, however, the two N-terminal mutants retained about 20% activity. Furthermore, these two pairs of mutants showed distinct binding abilities as to several general transcription factors. To obtain structural insights into the asymmetry, here we have analyzed the structures of the four cysteine mutants of the zinc ribbon domain by CD and NMR. All four mutants possessed a characteristic partially folded structure coordinating with a zinc atom, despite the imperfect set of cysteine-ligands. However, they equilibrated with several structures including the random coil structure. Unexpectedly, the two N-terminal mutants mainly equilibrated with the random coil structure, while the two C-terminal ones mainly equilibrated with folded structures. The characteristic structure formation of each mutant was reversible, which totally depended on the zinc binding.
* Present address: Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194.