Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions.
In the preceding paper [Choo, Y. & Klug, A. (1994) Proc. Natl. Acad. Sci. USA 91, 11163-11167], we showed how selections from a library of zinc fingers displayed on phage yielded fingers able to bind to a number of DNA triplets. Here, we describe a technique to deal efficiently with the converse problem--namely, the selection of a DNA binding site for a given zinc finger. This is done by screening against libraries of DNA triplet binding sites randomized in two positions but having one base fixed in the third position. The technique is applied here to determine the specificity of fingers previously selected by phage display. We find that some of these fingers are able to specify a unique base in each position of the cognate triplet. This is further illustrated by examples of fingers which can discriminate between closely related triplets as measured by their respective equilibrium dissociation constants. Comparing the amino acid sequences of fingers which specify a particular base in a triplet, we infer that in most instances, sequence-specific binding of zinc fingers to DNA can be achieved by using a small set of amino acid-nucleotide base contacts amenable to a code.
Medical Research Council, Laboratory of Molecular Biology, Cambridge, United Kingdom.
Y
Y
Choo
A
A
Klug
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry*; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors