Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation

Using hydroxylamine mutagenesis in vitro, mutations were introduced into a short DNA fragment containing the two overlapping promoters of the Escherichia coli galactose operon and the start of the first gal gene, galE. The mutagenised fragment was inserted into a lac expression plasmid. In such a vector, lac expression is controlled by the gal promoter region. Amongst eighteen candidates in which expression was reduced due to mutations in the gal fragment, twelve contained promoter mutations and six carried mutations that reduce the initiation of galE translation. The candidates in which promoter activity was reduced contained mutations affecting the promoter P1, which is dependent on the cyclic AMP-receptor protein complex (cAMP-CRP) for activation. All carried mutations in the sequence 5'GTGA3' at the CRP binding site. One of the twelve also contained a second mutation affecting the second promoter, P2, which normally functions in the absence of cAMP-CRP. Amongst the six candidates affecting galE translation, two contained a mutation that changes the initiator codon from AUG to AUA and almost completely suppresses galE expression. The mutations in the other four candidates affect the ribosome binding sequence, 5'GGAG3'. However, multiple mutations that abolish this sequence do not totally suppress galE expression, showing that there must be another way to guide ribosomes to the correct initiation site.     

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.     

Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the -35 region

A set of the lac promoter mutants that have varying lengths of the spacer between the CAP binding site and the -35 region was constructed. The mutants have the spacer length increased by five (I5 mutant), or eleven (I11) residues or decreased by eleven residues (D11). We also present a construction of the hybrid between the gal and lac promoters in which the CAP binding site and the -35 region of the gal promoter are fused to the lac -10 region. The promoter fragments were assembled through ligations of chemically synthesized oligodeoxynucleotides and cloned into a pBR322-derivative vector. The results of the in vivo assays of promoter activity show that the I11 mutation results in an active but weak promoter that can be stimulated by CAP, though to a lesser degree than the wild-type lac promoter. The other mutants exhibit no promoter activity. Since the insertion of 11-bp preserves the location of the CAP binding site on the same side on the DNA helix, the data demonstrate the importance of spatial alignment between the CAP binding site and the promoter. The fact that the gal::lac hybrid is inactive as a promoter indicates also that catabolite activation is a highly complex process in which the -35 and -10 regions cannot be easily exchanged between promoters.     

Kinetics of RNA polymerase-promoter complex formation: effects of nonspecific DNA-protein interactions.

The rates of formation of RNA polymerase-promoter open complexes at the galactose P2 and lactose UV5 promoters of E. coli were studied using polyacrylamide gels to separate the heparin-resistant complexes from unbound DNA. Both the apparent rate and extent of reaction at these promoters are inhibited at excess RNA polymerase. This inhibition, which can be relieved by the addition of non-promoter DNA, is interpreted to be the result of occlusion of the promoter site by nonspecifically bound polymerase. Additionally, biphasic kinetics are observed at both gal P2 and lac UV5, but not at the PR promoter of phage lambda. This behavior disappears when the concentration of RNA polymerase in the binding reaction is less than that of the promoter fragment. It is proposed that at excess enzyme nonspecifically bound polymerase molecules sliding along the DNA may "bump" closed complexes from the promoter site thereby reducing the rate of open complex formation. Kinetics mechanisms quantifying both the occlusion and bumping phenomena are presented.     

Ligand-modulated binding of a gene regulatory protein to DNA. Quantitative analysis of cyclic-AMP induced binding of CRP from Escherichia coli to non-specific and specific DNA targets

This paper describes a generally applicable method for quantitative investigation of ligand-dependent binding of a regulatory protein to its target DNA at equilibrium. It is used here to analyse the coupled binding equilibria of cAMP receptor protein from Escherichia coli K12 (CRP) with DNA and the physiological effector cAMP. In principle, the DNA binding parameters of CRP dimers with either one or two ligands bound are determinable in such an approach. The change of protein fluorescence was used to measure CRP binding to its recognition sequence in the lac control region and to non-specific DNA. Furthermore, the binding of cAMP to preformed CRP-DNA complexes was independently studied by equilibrium dialysis. The data were analysed using a simple interactive model for two intrinsically identical sites and site-site interactions. The intrinsic binding constant K and the co-operativity factor alpha for binding of cAMP to free CRP depend only slightly on salt concentration between 0.01 M and 0.2 M. In contrast, the affinity of cAMP for CRP pre-bound to non-specific DNA increases with the salt concentration and the co-operativity changes from positive to negative. This results from cation rebinding to the DNA lattice upon forming the cAMP-CRP-DNA complex from cAMP and the pre-formed CRP-DNA complex. The CRP-cAMP1 complex shows almost the same affinity for specific and non-specific DNA as the CRP-cAMP2 complex, and both displace the same number of cations. It is concluded that the allosteric activation of CRP is induced upon binding of the first cAMP. These results are used to estimate the occupation of the CRP site in the lac control region in relation to the cAMP concentration in vivo. Under physiological conditions the lac promoter is activated by the CRP dimer complexed with only one cAMP. Furthermore, a model for the differential activation of various genes expressed under catabolite repression is presented and discussed.     

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.