Arginine substituted for leucine at position 195 produces a cyclic AMP-independent form of the Escherichia coli cyclic AMP receptor protein.
Mutant forms (CRP*) of the Escherichia coli cAMP receptor protein (CRP) that activate CRP-dependent promoters in the absence of the normal allosteric effector (cAMP) have been described. A previous report (Harman, J. G., McKenney, K., and Peterkofsky, A. (1986) J. Biol. Chem. 261, 16332-16339) detailed the properties of three CRP* mutant proteins. One protein, 220 CRP, has amino acid substitutions at positions 127 and 170 and low CRP* activity in vivo. A second protein, 222 CRP, has the amino acid substitutions present in 220 CRP and a third substitution (arginine for leucine) at position 195. 222 CRP has high CRP* activity in vivo and high apparent affinity for lacP DNA relative to the 220 CRP in vitro. In this report, we evaluate the effect of a single amino acid substitution at position 195 (leucine to arginine) on CRP activity both in vivo and in vitro. Cells (cya delta crp delta/pJH8crpR195) containing R195 CRP were found to exhibit a CRP* phenotype, expressing a variety of CRP-dependent genes in the absence of added cAMP. R195 CRP exhibited both CRP* activity in vitro and increased apparent affinity for cAMP relative to wild-type CRP. CRP titration experiments performed using an in vitro lac transcription system suggest that the isolated substitution of arginine at position 195 does not confer on CRP the high lacP affinity that distinguishes the 220 and 222 forms of CRP. These findings lead us to the conclusion that the effects of multiple mutations in CRP can be both cumulative and interactive.
Harman
J G
JG
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Peterkofsky
A
A
McKenney
K
K
D001120:Arginine; D000242:Cyclic AMP / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis*; D007930:Leucine; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000032:analysis* / Q000235:genetics; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D011953:Receptors, Cyclic AMP; D001120:Arginine; D000242:Cyclic AMP; D007930:Leucine