Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein.
cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006460:Hemolysin Proteins; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013095:Spermidine / Q000494:pharmacology; D013696:Temperature; D014158:Transcription, Genetic / Q000187:drug effects; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; C039092:CAMP protein, Streptococcus; D006460:Hemolysin Proteins; D000242:Cyclic AMP; D001616:beta-Galactosidase; D013095:Spermidine