In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities

Takashi Sera, Peter G. Schultz

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'- A5T4T3G2C1G(1')C(2')A3A(4')T(5')-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14-Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.

Original languageEnglish
Pages (from-to)2920-2925
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number7
DOIs
Publication statusPublished - Apr 2 1996
Externally publishedYes

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Leucine Zippers
CCAAT-Enhancer-Binding Proteins
Basic Amino Acids
Asparagine
DNA
Mutant Proteins
Methionine
Libraries
Proteins
GAT

ASJC Scopus subject areas

  • General

Cite this

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abstract = "A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'- A5T4T3G2C1G(1')C(2')A3A(4')T(5')-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14-Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.",
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