Mechanism of CRP-mediated cya suppression in Escherichia coli

Abstract

Escherichia coli strain NCR30 contains a cya lesion and a second-site cya suppressor mutation that lies in the crp gene. NCR30 shows a pleiotropic phenotypic reversion to the wild-type state in expressing many operons that require the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex for positive control. In vivo 3-galactosidase synthesis in NCR30 was sensitive to glucose-mediated repression, which was relieved not only by cAMP but also by cyclic GMP and cyclic CMP. The CRP isolated from NCR30 differed from the protein isolated from wild-type E. coli in many respects. The mutant protein bound cAMP with four to five times greater affinity than wild-type CRP. Protease digestion studies indicated that native NCR30 CRP exists in the cAMP-CRP complex-like conformation. The protein conferred a degree of cAMP independence on the in vitro synthesis of P-galactosidase. In addition, the inherent positive control activity of the mutant protein in vitro was enhanced by those nucleotides that stimulate in vivo P-galactosidase synthesis in NCR30. The results of this study supported the conclusion that the crp allele of NCR30 codes for a protein having altered effector specificity yet capable of promoting positive control over catabolite-sensitive operons in the absence of an effector molecule.

Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP

“…reports (Dessein et aL, 1978;Takebe et aL, 1978;Alexander;1980;Melton et aL, 1981;Harman and Dobrogosz, 1983), confirming that catabolite repression can occur in crp* cells in the absence of cAMP.…”

Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.

“…Mutations in the crp gene which allow CRP to act in the absence of cAMP have been reported by several groups (Sanders and McGeoch, 1973;Dessein et al, 1978;Botsford and Drexler, 1978;Takebe etal., 1978;Alexander, 1980;Melton etal., 1981;Harman and Dobrogosz, 1983). The characteristics of such mutations are summarized in Table III.…”

“…These mutants can be divided into two types according to their response to cGMP. The crp* (Sanders and McGeoch, 1973;Dessein et al, 1978), crps (Takebe et al, 1978), sup (Alexander, 1980) and csm (Melton et al, 1981;Harman and Dobrogosz, 1983) are the first type whose protein products are activated by cGMP as well as cAMP and probably may have an increased affinity for cAMP. The crp*l, 2, 3, 4 and 6 described here belong to this type of mutation.…”

“…Such amino acid substitutions would favor the conformation responsible for the CRP* phenotype. Harman and Dobrogosz (1983) reported that their mutant CRP can activate lac expression without any small molecules in the cell-free transcription-translation system. Although the present work does not tell us whether CRP* require cofactors other than cAMP for activity, the striking similarity between their mutant crp (csm) and crp* described here suggest that most CRP* may be able to act without any effector molecules.…”

“…Although the native CRP absolutely requires cAMP to exert its regulatory function, a novel class of mutations in the crp gene produces an altered CRP partially independent of cAMP. Such mutations have been called crp* (Sanders and McGeoch, 1973;Dessein et al, 1978), crpi (Botsford and Drexler, 1978), crps (Takebe et al, 1978), sup (Alexander, 1983) and csm (Melton et al, 1981;Harman and Dobrogosz, 1983). To understand the molecular basis of these mutations, we isolated the crp* by in vitro mutagenesis of a plasmid containing the crp gene.…”