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

被引：19

作者：

Sera, T ^{[1
]}

Schultz, PG ^{[1
]}

机构：

[1] UNIV CALIF BERKELEY,DEPT CHEM,HOWARD HUGHES MED INST,BERKELEY,CA 94720

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1996年 / 93卷 / 07期

关键词：

D O I：

10.1073/pnas.93.7.2920

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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'-A(5)T(4)T(3)G(2)C(1)G(1')C(2')A(3)A(4')T(5')-3'. Mutants containing the sequence Leu(-18)Tyr(-15)Xaa(-14). Tyr(-11)Arg(-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.

引用

页码：2920 / 2925

页数：6

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