Study of the regulation of crp gene expression in Escherichia coli K12

The regulation of crp gene expression by CRP-cAMP complex was studied in E. coli strain by the crp-lac operon fusion. F'141 crp+ episome decreased 5-7 fold the high level of crp-lac expression in crp strains while F'141 crp episome had no effect. The hybrid plasmid pCAP2 crp+ with the intact crp gene did not affect the crp gene expression level in crp mutants, though they had acquired the Crp+ phenotype just as they did in F'141 crp+ presence. The F'141 crp+ and pCAP2 crp+ combination in crp mutants also resulted in decrease of the crp gene expression comparable to the registered in the presence of the F'141 crp+ plasmid. Similar repression occurred only in cya+ strains but not in cya strains. The crp gene is supposed to possess negative regulation by CRP-cAMP complex with a complementary factor also necessary. The latter is evidently located in an E. coli chromosome site overlapped by F'141 episome.     

Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation.

The three-dimensional model of the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP) shows that several amino acids are involved as chemical contacts for binding cAMP. We have constructed and characterized mutants at four of these positions, E72, R82, S83, and R123. The mutations were made in wild-type crp as well as a cAMP-independent crp, crp*. The activities of the mutant proteins were characterized in vivo for their ability to activate the lac operon. These results provide genetic evidence to support that E72 and R82 are essential and S83 and R123 are important in the activation of CRP by cAMP.     

Sites of allosteric shift in the structure of the cyclic AMP receptor protein

We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression. Cyclic GMP, normally an ineffective activator of CRP+, can stimulate these mutant CRP*s to permit greater lac expression in vivo. Cyclic AMP binding to the amino-terminal domain of CRP+ induces an allosteric transition that changes the DNA-binding property of the carboxy domain. The CRP* phenotype is caused by substitution of amino acids with bulkier side chains in the D alpha-helix of the protein's carboxy domain, near the hinge connecting the two domains. These results are consistent with a model in which the mutant CRP*s assume, in part, a conformation normally evoked only by cAMP binding: one in which the relative orientation of the C, D, and F alpha-helices is altered. We define precisely the amino acids of these alpha-helices that interact to cause the allosteric shift.     

大肠杆菌棉子糖操纵子α—半乳糖苷酶表达的调节控制

The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS)