title: Specific DNA binding of the cAMP receptor protein within the lac operon stabilizes double-stranded DNA in the presence of cAMP.
abstract: The effects of varying amounts of cAMP receptor protein (CRP) in the presence and absence of cAMP on the melting and differential melting curves of a 301-bp fragment containing the lac control region in 5 mM Na+ have been investigated. The native 301-bp fragment consists of three cooperatively melting thermalites. At 5 mM Na+, thermalite I (155 bp) has a Tm of 66.4 degrees C and the melting transitions of thermalites II (81 bp) and III (65 bp) are superimposed with a Tm of 61.9 degrees C. The specific DNA target site for CRP and the lac promotor are located within thermalite II. CRP alone exerts no specific effects on the melting of the 301-bp fragment, non-specific DNA binding of CRP resulting in a progressive stabilization of the double-stranded DNA by increasing the number of base pairs melting at a higher Tm in a non-cooperative transition. The cAMP-CRP complex, however, exerts a specific effect with a region of approximately 36 bp, comprising the specific CRP binding site and a neighbouring region of DNA, being stabilized. The appearance of this new cooperatively melting region, known as thermalite IV, is associated with a corresponding decrease in the area of thermalites II/III. The Tm of thermalite IV is 64.4 degrees C, 2.5 degrees C higher than that of thermalites II/III. With two or more cAMP-CRP complexes bound per 301-bp fragment, the stabilization also affects the remaining 110 bp now making up thermalites II/III whose Tm is increased by 1 degrees C to 62.9 degrees C. The implications of these findings for various models of the mode of action of the cAMP-CRP complex are discussed.
authors.affiliation: Division of Molecular Pharmacology, National Institute for Medical Research, London, UK.
authors.forename: B
authors.initials: B
authors.lastname: Unger
authors.forename: G M
authors.initials: GM
authors.lastname: Clore
authors.forename: A M
authors.initials: AM
authors.lastname: Gronenborn
authors.forename: W
authors.initials: W
authors.lastname: Hillen
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007763:Lac Operon
chemical_list: D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D004247:DNA; D000242:Cyclic AMP