ABSTRACT
The allosteric mechanism by which the gene expression regulatory protein AraC regulates its DNA-binding activity is shown to be portable by grafting it to beta-galactosidase, generating an arabinose-regulated beta-galactosidase. A portion of the alpha-peptide sequence that complements the activity of alpha-acceptor beta-galactosidase was inserted into a nonessential region of the regulatory peptidyl arm of AraC protein. Arabinose, which regulates the position of the arm in AraC protein now regulates the availability of the alpha-peptide to alpha-acceptor beta-galactosidase, thereby modulating its activity in response to arabinose.
Subject(s)
Bacterial Proteins/chemistry , Repressor Proteins/chemistry , Transcription Factors/chemistry , Allosteric Regulation , AraC Transcription Factor , Arabinose/metabolism , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Structure-Activity Relationship , Transcription Factors/metabolism , beta-Galactosidase/chemistry , beta-Galactosidase/metabolismABSTRACT
The previously isolated hemiplegic, induction-negative, repression-positive mutants, H80R and Y82C, were found to be defective in the binding of arabinose. Randomization of other residues close to arabinose in the three-dimensional structure of AraC or that make strong interactions with arabinose yielded induction-negative, repression-positive mutants. The induction and repression properties of mutants obtained by randomizing individual residues of the N-terminal arm of AraC allowed identification of the domain with which that residue very likely makes its predominant interactions. Residues 8-14 of the arm appear to make their predominant interaction with the DNA-binding domain. Although the side-chain of residue 15 interacts directly with arabinose bound to the N-terminal dimerization domain, the properties of mutant F15L indicate that this mutation increases the affinity of the arm for the DNA-binding domain.
