Isolation of a functional exuR-repressor-beta-galactosidase hybrid protein by use of in vitro gene fusions.
The exuR regulatory gene of Escherichia coli codes for a repressor molecule that controls negatively the expression of the exu regulon genes involved in the hexuronate degradation. The exuR gene was fused to the lacZ gene in vitro on plasmid cloning vectors developed by Casadaban et al. [J. Bacteriol. 143 (1980) 971-980]. The exuR-lacZ fusion gene allows the production of hybrid molecules possessing in a single polypeptide beta-galactosidase activity as well as the capacity to bind in vivo specifically to the operators of the exu operons. However, the affinity of ExuR hybrid protein for the exu operators seems to be reduced in comparison with the native repressor since, even overproduced in the cell, the chimeric polypeptide was not able to completely repress the expression of the exu operons. The other functions of the ExuR repressor, i.e., repression of the uxuAB operon and inactivation by the inducers, were not retained in the chimeric protein. This particular chimaera of Mr about 120 000 was purified by chromatography on phosphocellulose from strains carrying the fusion plasmids. The purified polypeptide was able to repress in vitro the synthesis of an exu gene product in a cell-free transcription and translation system. The implications of the repressor activity in the chimeric protein are discussed. The NH2-terminus of ExuR repressor probably recognizes the exu operators specifically, and this region represents less than 20% of the entire ExuR molecule.
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003470:Culture Media; D004269:DNA, Bacterial / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D010957:Plasmids; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D005696:Galactosidases; D001616:beta-Galactosidase