Constitutive mutations in the Escherichia coli AraC protein.
The Escherichia coli AraC protein represses and induces the araBAD operon in response to the absence or presence of l-arabinose. Constitutive mutations in the AraC gene no longer require the presence of l-arabinose to convert AraC from its repressing to its inducing state. Such mutations were isolated directly by virtue of their constitutivity or by their resistance to the nonmetabolizable arabinose analog, d-fucose. The majority of the constitutive mutations lie within the same residues of the N-terminal regulatory arm of AraC. Two, however, were found in the core of the dimerization domain. As predicted by the light switch mechanism of AraC, constitutive mutations increase the susceptibility of the N-terminal arms to digestion by trypsin or chymotrypsin, suggesting that these mutations weaken or disrupt the arm structure required for repression by AraC. Fluorescence, circular dichroism, and cysteine reactivity measurements show that the constitutive mutations in the core of the dimerization domain lead to a weakening of the support for the arms and reduce the stability of the minus-arabinose arm structure. These mutations also weaken the interaction between the two-helix bundle and the beta-barrel subdomains of the dimerization domain and reduce the structural stability of the beta-barrels.
Dirla
Stephanie
S
Biology Department, Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218, USA. schleif@jhu.edu
Chien
John Yeh-Heng
JY
Schleif
Robert
R
D019943:Amino Acid Substitution / Q000235:genetics; D051777:AraC Transcription Factor / Q000096:biosynthesis* / Q000235:genetics*; D001089:Arabinose / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005643:Fucose / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008954:Models, Biological; D008958:Models, Molecular; D020125:Mutation, Missense; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D029968:Escherichia coli Proteins; D005643:Fucose; D001089:Arabinose; D002918:Chymotrypsin; D014357:Trypsin