Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.     

Protein-DNA cross-linking at the lac promoter.

We report the results of photo-cross-linking of RNA polymerase and the cyclic AMP receptor protein (CRP) to the lac UV5 promoter region carried on either a linear fragment or a supercoiled plasmid. We have devised a protocol that allows the localisation of bases in contact with the protein. RNA polymerase makes contacts within the -10 and -35 regions of the promoter, essentially on the non-template strand. The CRP contact points found in a binary complex are affected by the formation of the ternary complex containing RNA polymerase. Supercoiling has no effect on the position of contacts in any of the complexes. These conclusions were derived from experiments performed using a generally applicable, non-interfering technique that reveals direct contacts between proteins and nucleic acids in nucleoprotein complexes.     

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.     

Is DNA unwound by the cyclic AMP receptor protein?

Superhelical pBR 322 derivatives have been relaxed by eukaryotic topoisomerase I in the presence or in the absence of E. coli cyclic AMP receptor protein (CRP) and of cyclic AMP (cAMP). CRP alone, or cAMP alone do not affect the average linking number of the distribution of the relaxed topoisomers. Hence, they do not unwind the template. In the presence of cAMP, CRP induces a small unwinding. The extent of this unwinding is barely modified when the relaxation is carried out on a similar vector plasmid where the CRP binding site of the lac or of the gal operon has been inserted. Under these conditions, we checked that CRP occupies the lactose control site and that upon addition of RNA polymerase, the corresponding promoter is readily activated. These findings are difficult to reconcile with the proposal that activation of these promoters results from the binding of the CRP-cAMP complex to left-handed DNA sequences.     

Tandem CRP binding sites in the deo operon of Escherichia coli K-12

The locations of DNA binding by the cyclic AMP receptor protein (CRP) in the deo operon of Escherichia coli have been determined by the DNase I footprinting procedure. Two high affinity sites were found around positions -35 and -90, preceding the second deo promoter. In vitro data on induction of gene fusions that join different parts of the deoP -2 regulatory region to the lac genes suggest that: (1) both CRP binding sites are needed for high expression from the deoP -2 region; and (2) negative regulation by the cytR repressor is accomplished by preventing the cAMP-CRP complex from binding to the second target.     

Mutations in the promoter regions of the malEFG and malK-lamB operons of Escherichia coli K12

The malB region of Escherichia coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. Expression of the malB operons is under the positive control of the malT gene product (MalT) and maltose and of the crp gene product (CRP) and cyclic AMP. Strains in which the lac genes have been fused to malE or malK are unable to use lactose as carbon source if they have been deleted for malT or crp. Mutations in the malB region allowing such fusion strains to grow on lactose have been isolated. These and previously isolated mutations were genetically characterized. As regards the malEp promoter mutations, malEp9, malEp1 and malEp6 create new promoters that are MalT and CRP independent. malEp9 and malEp1 change residues -1 and -2, respectively, of malEp without altering its activity. malEp6 duplicates six base-pairs between residues -22 and -23. malEp3 improves the -10 region hexamer. malEp5 deletes residues -29 to -62. It creates a new promoter that is MalT independent, CRP dependent, likely by fusing together functional regions of malEp that are normally apart. malEp5 also reduces the expression of malK-lamB, suggesting the existence of a link between the malEp and malKp promoters. As regards the malKp mutations, malKp6 changes residue -81 of malKp without altering its activity. It creates a new promoter, which is MalT independent, CRP dependent, likely by using a pre-existing cyclic AMP/CRP binding site. malKp102 changes residue -36, two bases upstream of the -35 region hexamer. It decreases the activity of malKp by at least four orders of magnitude and likely alters the MalT binding site. These results are discussed in terms of regulatory interactions within the malB control region.     

Cyclic AMP receptor proteins interacts with lactose operator DNA

The wildtype lac promoter-operator region of E. coli was reinvestigated for cyclic AMP receptor protein (CRP) binding sites using the 'footprinting' method. Two distinct interaction sites were found, one in the -60 base pair region, which has previously been described, and a second one covering the lac operator. These two sites are similar in extent, orientation and the characteristic pattern of CRP-induced DNaseI enhanced cutting sites.