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

Journal Article · Tue Apr 02 04:00:00 EST 1996 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.93.7.2920· OSTI ID:258595

Sera, T; Schultz, P G ^[1]

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1. Univ. of California, Berkeley, CA (United States)

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{sup {minus}18}, Ala{sup {minus}15}, Val{sup {minus}14}, Ser{sup {minus}11}, And Arg{sup {minus}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{prime}-A{sup 5}T{sup 4}T{sup 3}G{sup 2}C{sup 1}G{sup 1{prime}}C{sup 2{prime}}A{sup 3}A{sup 4{prime}}T{sup 5{prime}}-3{prime}. Mutants containing the sequence Leu{sup {minus}18}Tyr{sup {minus}15}Xaa{sup {minus}14}-Tyr{sup {minus}11}Arg{sup {minus}10}, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5{prime}-ACTCYCGY{prime}GAT-3{prime} (Xaa=asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5{prime}-ATTACGTAAT-3{prime}, 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. 28 refs., 1 fig., 4 tabs.

• DOE Contract Number: AC03-76SF00098
• OSTI ID: 258595
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 7 Vol. 93; ISSN 0027-8424; ISSN PNASA6
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
BIOCHEMISTRY
DNA
IN VIVO
LEUCINE
MUTATIONS
PROTEINS
SPECIFICITY
TRANSCRIPTION