In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator.
Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.
Khoury
A M
AM
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
Nick
H S
HS
Lu
P
P
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon; D007786:Lactose Factors; D008745:Methylation; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis
D004269:DNA, Bacterial; D012097:Repressor Proteins; D001616:beta-Galactosidase