Protein stitchery: design of a protein for selective binding to a specific DNA sequence.
We present a general strategy for designing proteins to recognize DNA sequences and illustrate this with an example based on the "Y-shaped scissors grip" model for leucine-zipper gene-regulatory proteins. The designed protein is formed from two copies, in tandem, of the basic (DNA binding) region of v-Jun. These copies are coupled through a tripeptide to yield a "dimer" expected to recognize the sequence TCATCGATGA (the v-Jun-v-Jun homodimer recognizes ATGACTCAT). We synthesized the protein and oligonucleotides containing the proposed binding sites and used gel-retardation assays and DNase I footprinting to establish that the dimer binds specifically to the DNA sequence TCATCGATGA but does not bind to the wild-type DNA sequences, nor to oligonucleotides in which the recognition half-site is modified by single-base changes. These results also provide strong support for the Y-shaped scissors grip model for binding of leucine-zipper proteins.
Park
C
C
Materials and Molecular Simulation Center, Beckman Institute (139-74), Pasadena, CA.
Campbell
J L
JL
Goddard
W A
WA
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D016759:Oncogene Protein p65(gag-jun) / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D016759:Oncogene Protein p65(gag-jun); D004247:DNA; D003850:Deoxyribonuclease I