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.
D000911:Antibodies, Monoclonal / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011953:Receptors, Cyclic AMP / Q000276:immunology / Q000502:physiology*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D000939:Epitopes; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I