MONOCLONAL ANTIBODIES DIRECTED AGAINST THE CYCLIC-AMP RECEPTOR PROTEIN OF ESCHERICHIA COLI AS PROBES OF STRUCTURE AND FUNCTION.
Item
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Title
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MONOCLONAL ANTIBODIES DIRECTED AGAINST THE CYCLIC-AMP RECEPTOR PROTEIN OF ESCHERICHIA COLI AS PROBES OF STRUCTURE AND FUNCTION.
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Identifier
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AAI8713774
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identifier
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8713774
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Creator
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LI, XIAO-MIAO.
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Contributor
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Joseph S. Krakow
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Monoclonal antibodies (mAb) against the E. coli cAMP receptor protein (CRP) have been isolated and characterized. The anti CRP mAbs fall into two groups: Class I mAbs bind only to native but not urea denatured CRP. Class II mAbs bind equally well to both native and urea denatured CRP. The locations of the antigenic determinants of the mAbs have been studied. All Class I mAbs studied are located in the C-terminal domain of CRP and all Class II mAbs studied are located in the N-terminal domain. Class II mAb 64D1 strongly inhibits cAMP binding by CRP by altering the conformation of CRP and consequently also inhibits binding of CRP to the lac promoter and abortive initiation by RNA polymerase. CRP in the preformed open promoter complex is protected from attack by mAB 64D1 indicating that binding of RNA polymerase stabilizes the cAMP-CRP complex. The antigenic determinants of mAb 66C3 and mAb 63B2 are located at the hinge region of CRP joining the large and small domains. Both mAbs are strong inhibitors of transcription initiation from the lac promoter; mAb 63B2 inhibits DNA binding by CRP while mAb 66C3 enhances this binding. DNase I footprinting indicates that cAMP-CRP-mAb 66C3 binds to CRP Site 1 (-50 to -70 bp) and also to CRP Site 2 (-10 to +10 bp) which overlaps part of the RNA polymerase binding site. 50% of the preformed open promoter complex resists attack by mAb 66C3. The results imply that two states of CRP in the open promoter complex might exist due to the asymmetry in the cAMP-CRP complex. Protein-protein interactions between CRP and RNA polymerase are also suggested by a comparison of the DNase I footprint patterns of lac P('+) and lac L8UV5 complexes.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
