Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA.
The short sequence motif named 'zinc finger', first recognized repeated in tandem in the Xenopus transcription factor IIIA (TFIIIA), is also found in the yeast transcriptional activator SWI5 (ref. 3) and many other regulator proteins. Embedded in the 709-amino-acid polypeptide chain of SWI5 are three tandemly repeated zinc-finger motifs. Because the zinc fingers of TFIIIA are known to bind to DNA, it is probable that in the case of SWI5 these finger motifs also play an important, but not necessarily exclusive, role in the sequence-specific binding of the protein to DNA. To test this prediction we have expressed the 89-amino-acid sequence of the domain containing the three zinc fingers of SWI5 in Escherichia coli as a cleavable fusion protein, purified under denaturing conditions and folded in vitro. This experimental approach allows us to study directly both the metal requirement and DNA-binding properties of the isolated polypeptide. We find that zinc is required for specific DNA recognition and, most significantly, DNaseI protection studies show that the isolated three-fingered domain is sufficient for sequence-specific binding to DNA.
Nagai
K
K
MRC Laboratory of Molecular Biology, Cambridge, UK.
Nakaseko
Y
Y
Nasmyth
K
K
Rhodes
D
D
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000235:genetics; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics*; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015032:Zinc
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D008667:Metalloproteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D004262:DNA Restriction Enzymes; C037725:SCEI protein, S cerevisiae; D003850:Deoxyribonuclease I; D015252:Deoxyribonucleases, Type II Site-Specific; D015032:Zinc; D003488:Cyanogen Bromide