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More fingers in hand
Cell 444
this focus on infected cells rather than bystanders that makes the compartmentalization model so appealing, since it provides a means to restrict the potent class I-restricted cytotoxic T effector mechanism to the appropriate Cell, Vol. 54, 444, August 12, 1988, Copyright
target ceils, rather than permitting exogenously acquired antigen to convert the wide range of class l-positive somatic cells into the innocent victims of an individual’s own immune defenses.
0 1988 by Cell Press
Letter to the Editor More Fingers
in Hand
Since its discovery in Xenopus transcription factor IIIA (TFIIIA), the “zinc finger” structural motif has been invoked in numerous nucleic acid binding proteins. In the case of TFIIIA, it was proposed that each of nine zinc fingers is comprised of a pair of cysteines and a pair of histidines that form a tetrahedral coordination complex with a zinc ion, thus creating a peptide loop that interacts specifically with target DNA (Miller et al., EMBO J. 4, 1609-1614, 1985). The Zn-coordination scheme has been confirmed by extended X-ray absorption fine structure (EXAFS) (Diakun et al., Nature 324, 698-699, 1986) and visiblelight (Frankel et al., PNAS 84, 4841-4845, 1987) spectroscopies. Moreover, yeast transcription factor SW15 and human factor Spl, which both have finger motifs closely related to TFIIIA, require zinc for specific DNA binding (Nagai et al., Nature 332,284-286,1988; Kadonaga et al., Cell 57, 1079-1090,1987). In principle, other combinations of cysteines and histidines could also serve as zinc coordination sites, and some have been inferred by inspection of sequences of other DNA binding proteins (for reviews, see Klug and Rhodes, Trends Biochem. Sci. 72,464-469, 1987; and Evans and Hollenberg, Cell 52, l-3, 1988). At present, the structure of the zinc finger remains a matter of conjecture (Berg, PNAS 85,99-102, 1988). Consequently, the test for classification as a finger protein has commonly been satisfied merely by demonstration of the requisite arrangement of Cys and His residues. Frankel and Pabo (Cell 53,675,1988) have recently questioned the validity of this sequence criterion for identification of zinc fingers. Furthermore, they propose that only proteins with the Cys-X2d-Cys-&-Phe-X-Xss-Leu-Xs-His-Xw-His motif of TFIIIA should qualify as bona fide zinc finger proteins; specifically, they suggest that the zinc finger-like all-cysteine motif found in the DNA binding domains of steroid receptors and other members of the nuclear receptor superfamily may in reality be structurally unrelated to the true zinc fingers of TFIIIA. We have made several experimental observations relevant to these points. Using the DNA binding domain of the glucocorticoid receptor, which we expressed in E. coli and purified to homogeneity (Freedman et al., Nature, in press), we showed by spectroscopic methods that the protein reversibly ligates two Zn(ll) ions, and by functional assays that the metal ions are required for specific DNA binding and for stable folding. By EXAFS and visible-light spectroscopies, we found that each Zn atom is coordi-
nated tetrahedrally by four cysteines. Thus, we conclude that the receptor, like TFIIIA, coordinates metal in fingerlike structures that are essential for sequence-specific recognition of DNA. Frankel and Pabo (op. cit.) point out that two receptor molecules might dimerize by coordinating a common metal ion through bridging cysteine side chains from each protein. Such an atomic arrangement would be similar to that of metallothionein and has been proposed to explain dimerization of the HIV Tat protein (Frankel et al., Science 240,70-73,1988). Our observations appear to rule out this scheme for the glucocorticoid receptor. First, ultracentrifugation and gel permeation chromatography demonstrate that the purified receptor DNA binding domain is a monomer in solution (L.P.F., unpublished). Second, coordination of two zinc ions, each by four unique cysteines, would leave two cysteines free, one in each “fingertip.” As predicted, spectrophotometric titration of the receptor DNA binding domain with a mercurial reagent reveals two reactive sulfhydryls per protein molecule (Freedman et al., op. cit.). Sharing of a single cysteine by two zinc ions, as in metallothionein, would yield at least three free cysteine residues. Certainly the mere presence of appropriately placed cysteines or histidines should not suffice to identify zinc finger proteins. However, the proposal that a true “zinc finger” should correspond to the particular motif found in TFIIIA seems to us to be unduly restrictive. Our results suggest strongly that a member of the nuclear receptor family contains such fingers despite their lack of similarity to the TFIIIA consensus. Clearly, definitive comparisons await information on three-dimensional structure, but it seems likely that sequence differences among the zinc fingers may reflect variations on a common structural theme, perhaps generating diversity of DNA sequence recognition and associated functions among the members of this class of proteins. Leonard P. Freedman,” Keith R. Yamamoto: Ben F. Luisi,t and Paul 8. Siglert ‘Department of Biochemistry and Biophysics University of California, San Francisco San Francisco, California 94143-0448 *Department of Biochemistry and Molecular Biology The University ‘of Chicago Chicago, Illinois 60637
this focus on infected cells rather than bystanders that makes the compartmentalization model so appealing, since it provides a means to restrict the potent class I-restricted cytotoxic T effector mechanism to the appropriate Cell, Vol. 54, 444, August 12, 1988, Copyright
target ceils, rather than permitting exogenously acquired antigen to convert the wide range of class l-positive somatic cells into the innocent victims of an individual’s own immune defenses.
0 1988 by Cell Press
Letter to the Editor More Fingers
in Hand
Since its discovery in Xenopus transcription factor IIIA (TFIIIA), the “zinc finger” structural motif has been invoked in numerous nucleic acid binding proteins. In the case of TFIIIA, it was proposed that each of nine zinc fingers is comprised of a pair of cysteines and a pair of histidines that form a tetrahedral coordination complex with a zinc ion, thus creating a peptide loop that interacts specifically with target DNA (Miller et al., EMBO J. 4, 1609-1614, 1985). The Zn-coordination scheme has been confirmed by extended X-ray absorption fine structure (EXAFS) (Diakun et al., Nature 324, 698-699, 1986) and visiblelight (Frankel et al., PNAS 84, 4841-4845, 1987) spectroscopies. Moreover, yeast transcription factor SW15 and human factor Spl, which both have finger motifs closely related to TFIIIA, require zinc for specific DNA binding (Nagai et al., Nature 332,284-286,1988; Kadonaga et al., Cell 57, 1079-1090,1987). In principle, other combinations of cysteines and histidines could also serve as zinc coordination sites, and some have been inferred by inspection of sequences of other DNA binding proteins (for reviews, see Klug and Rhodes, Trends Biochem. Sci. 72,464-469, 1987; and Evans and Hollenberg, Cell 52, l-3, 1988). At present, the structure of the zinc finger remains a matter of conjecture (Berg, PNAS 85,99-102, 1988). Consequently, the test for classification as a finger protein has commonly been satisfied merely by demonstration of the requisite arrangement of Cys and His residues. Frankel and Pabo (Cell 53,675,1988) have recently questioned the validity of this sequence criterion for identification of zinc fingers. Furthermore, they propose that only proteins with the Cys-X2d-Cys-&-Phe-X-Xss-Leu-Xs-His-Xw-His motif of TFIIIA should qualify as bona fide zinc finger proteins; specifically, they suggest that the zinc finger-like all-cysteine motif found in the DNA binding domains of steroid receptors and other members of the nuclear receptor superfamily may in reality be structurally unrelated to the true zinc fingers of TFIIIA. We have made several experimental observations relevant to these points. Using the DNA binding domain of the glucocorticoid receptor, which we expressed in E. coli and purified to homogeneity (Freedman et al., Nature, in press), we showed by spectroscopic methods that the protein reversibly ligates two Zn(ll) ions, and by functional assays that the metal ions are required for specific DNA binding and for stable folding. By EXAFS and visible-light spectroscopies, we found that each Zn atom is coordi-
nated tetrahedrally by four cysteines. Thus, we conclude that the receptor, like TFIIIA, coordinates metal in fingerlike structures that are essential for sequence-specific recognition of DNA. Frankel and Pabo (op. cit.) point out that two receptor molecules might dimerize by coordinating a common metal ion through bridging cysteine side chains from each protein. Such an atomic arrangement would be similar to that of metallothionein and has been proposed to explain dimerization of the HIV Tat protein (Frankel et al., Science 240,70-73,1988). Our observations appear to rule out this scheme for the glucocorticoid receptor. First, ultracentrifugation and gel permeation chromatography demonstrate that the purified receptor DNA binding domain is a monomer in solution (L.P.F., unpublished). Second, coordination of two zinc ions, each by four unique cysteines, would leave two cysteines free, one in each “fingertip.” As predicted, spectrophotometric titration of the receptor DNA binding domain with a mercurial reagent reveals two reactive sulfhydryls per protein molecule (Freedman et al., op. cit.). Sharing of a single cysteine by two zinc ions, as in metallothionein, would yield at least three free cysteine residues. Certainly the mere presence of appropriately placed cysteines or histidines should not suffice to identify zinc finger proteins. However, the proposal that a true “zinc finger” should correspond to the particular motif found in TFIIIA seems to us to be unduly restrictive. Our results suggest strongly that a member of the nuclear receptor family contains such fingers despite their lack of similarity to the TFIIIA consensus. Clearly, definitive comparisons await information on three-dimensional structure, but it seems likely that sequence differences among the zinc fingers may reflect variations on a common structural theme, perhaps generating diversity of DNA sequence recognition and associated functions among the members of this class of proteins. Leonard P. Freedman,” Keith R. Yamamoto: Ben F. Luisi,t and Paul 8. Siglert ‘Department of Biochemistry and Biophysics University of California, San Francisco San Francisco, California 94143-0448 *Department of Biochemistry and Molecular Biology The University ‘of Chicago Chicago, Illinois 60637