PMID:1905359
| Citation |
Khoury, AM, Nick, HS and Lu, P (1991) In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator. J. Mol. Biol. 219:623-34 |
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| Abstract |
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. |
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| Keywords |
Base Sequence; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Lac Operon; Lactose Factors; Methylation; Molecular Sequence Data; Operator Regions, Genetic; Promoter Regions, Genetic; Repressor Proteins/genetics; Repressor Proteins/metabolism; beta-Galactosidase/biosynthesis |
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