Unger B, Clore G M, Gronenborn A M, Hillen W
Division of Molecular Pharmacology, National Institute for Medical Research, London, UK.
EMBO J. 1983;2(2):289-93. doi: 10.1002/j.1460-2075.1983.tb01419.x.
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.
研究了在存在和不存在环磷酸腺苷（cAMP）的情况下，不同量的cAMP受体蛋白（CRP）对含乳糖操纵子控制区的301碱基对片段在5 mM Na⁺中的解链和差示解链曲线的影响。天然的301碱基对片段由三个协同解链的热区组成。在5 mM Na⁺时，热区I（155碱基对）的解链温度（Tm）为66.4℃，热区II（81碱基对）和III（65碱基对）的解链转变叠加，Tm为61.9℃。CRP的特异性DNA靶位点和乳糖启动子位于热区II内。单独的CRP对301碱基对片段的解链没有特异性影响，CRP的非特异性DNA结合通过增加在较高Tm下非协同转变时解链的碱基对数，导致双链DNA逐渐稳定。然而，cAMP-CRP复合物对约36碱基对的区域有特异性影响，该区域包括特异性CRP结合位点和相邻的DNA区域，使其稳定。这个新的协同解链区域，即热区IV的出现，与热区II/III的面积相应减小有关。热区IV的Tm为64.4℃，比热区II/III高2.5℃。每301碱基对片段结合两个或更多的cAMP-CRP复合物时，这种稳定作用也会影响现在构成热区II/III的剩余110碱基对，其Tm增加1℃至62.9℃。讨论了这些发现对cAMP-CRP复合物作用模式的各种模型的意义。
