In vitro evolution of the DNA binding sites of Escherichia coli methionine repressor, MetJ

被引:39
作者
He, YY
Stockley, PG
Gold, L
机构
[1] UNIV COLORADO,DEPT MOLEC CELLULAR & DEV BIOL,BOULDER,CO 80309
[2] UNIV LEEDS,DEPT GENET,LEEDS LS2 9JT,W YORKSHIRE,ENGLAND
关键词
SELEX; E. coli MetJ repressor; DNA ligand; gel retardation; dissociation constant;
D O I
10.1006/jmbi.1996.0006
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The SELEX procedure was used to study the recognition between the E. coli methionine repressor (MetJ) and its DNA binding sites. DNA ligands with high affinity for either the hole-repressor or ape-repressor were isolated from a pool of molecules randomized over 20 base-pairs. Among 90 DNA ligands selected by hole-repressor binding, roughly 90% contain variations of two tandem, perfect eight base-pair Met-boxes, which are the consensus deduced from natural met operators. Base-pairs that are important, for specific interactions with the protein are highly conserved. The data also reveal the importance of the non-contacted operator base-pairs in facilitating the conformational changes in the operator which must occur for repressor binding. There are also effects due to the sequences of the base-pairs immediately flanking the operator site. DNA ligands selected by apo-repressor share a very similar, but not identical, consensus with that selected by hole-repressor, suggesting that the corepressor does not greatly alter the specificity of repressor binding. (C) 1996 Academic Press Limited
引用
收藏
页码:55 / 66
页数:12
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