Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter

The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.     

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities

Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex. To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM. The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS). Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration. At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein. At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence. These results show unequivocally that there are at least three conformational states of the protein. The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration. Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes. The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique. At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased. This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.     

Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli

The environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercoiling. It is believed that protein-induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP-CRP on algD promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non-responsive to glucose repression. The involvement of cAMP-CRP complex in the activation of the algD promoter in E. coli has been demonstrated directly through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP-like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa.     

Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters

Polyacrylamide gel electrophoresis has been used to visualise and quantitate complexes between the Escherichia coli cyclic AMP receptor protein (CRP) and DNA fragments containing the promoter region of either the E. coli galactose or lactose operons. We show that, although CRP binding to the gal fragment is weaker than binding to the lac fragment, in each case, stable complexes are formed between one dimer of CRP and one molecule of DNA. We have examined the effects of a series of deletions and point mutations in the gal promoter region on CRP binding. From the position of deletions and mutations which prevent the formation of stable complexes, we deduce the location and extent of the sequence at the CRP binding site. We show that it covers approximately the same length of sequence as the binding site at the lac promoter. Unlike the lac site, the gal site contains no palindromic sequence. We discuss the importance of symmetry in the sequence at CRP binding sites and the validity of CRP binding consensus sequences which have been proposed.     

Identification of protein S 1 at the messenger RNA binding site of the Escherichia coli ribosome

Poly-4-thiouridylic acid acts as messenger RNA for polyphenylalanine synthesis in an $\text{in vitro}$ protein synthesizing system. When a complex consisting of ribosomes, poly-4-thiouridylic acid and Phe-tRNA is irradiated at 300 to 400 nm, covalent bonds between this messenger RNA and protein S 1 are formed.