Department of Biochemistry, Faculty of Medicine, Cumhuriyet University, 58140, Sivas, Turkey.
Mol Biol Rep. 2012 Apr;39(4):4261-5. doi: 10.1007/s11033-011-1212-5. Epub 2011 Jul 21.
Lac operon transcription activation through CRP dimer depends on cAMP second messenger. The formation of CRP homodimers is mediated by protein-protein interactions between the monomers. Cyclic AMP ligand binding brings CRP dimer to an active state via conformational changes. Molecular modeling studies in our lab showed the importance of monomer in transcription activation through its pre-existing conformational state. Until now CRP experiments were carried out at protein concentrations higher than that of CRP dimer dissociation value making all CRP monomers dimer and ignore the importance of CRP monomer in allosteric activation. Labeling CRP monomers with fluorophores exterminate using excess protein concentration and allow monitoring CRP monomer behavior. CRP monomer exchange accelerates in the presence of non specific DNA whereas the exchange is inhibited in the presence of specific DNA and cAMP ligand. Degree of subunit exchange depends on the stability of CRP dimer. Cyclic AMP forms a single molecule from two monomers and addition of specific DNA further stabilizes CRP dimer and decreases monomer exchange. On the other hand, addition of non specific DNA increases CRP monomer exchange and may explain the mechanism of CRP monomer removal and dissociation of CRP dimer:cAMP:DNA complex. The exchange behavior of CRP in the presence of different factors implies importance of monomer in transcription complex association and dissociation.
Lac 操纵子转录激活通过 CRP 二聚体依赖于 cAMP 第二信使。CRP 同源二聚体的形成是通过单体之间的蛋白质-蛋白质相互作用介导的。环腺苷酸配体结合通过构象变化将 CRP 二聚体转化为活性状态。我们实验室的分子建模研究表明，单体通过其预先存在的构象状态在转录激活中的重要性。到目前为止，CRP 实验都是在高于 CRP 二聚体解离值的蛋白浓度下进行的，这使得所有 CRP 单体都二聚化，并忽略了 CRP 单体在别构激活中的重要性。用荧光标记 CRP 单体可以消除过量蛋白浓度的影响，并允许监测 CRP 单体的行为。在非特异性 DNA 存在下，CRP 单体的交换加速，而在特异性 DNA 和 cAMP 配体存在下，交换被抑制。亚基交换的程度取决于 CRP 二聚体的稳定性。环腺苷酸由两个单体形成一个分子，特异性 DNA 的加入进一步稳定 CRP 二聚体并减少单体交换。另一方面，非特异性 DNA 的加入增加了 CRP 单体的交换，这可能解释了 CRP 单体的去除和 CRP 二聚体:cAMP:DNA 复合物的解离机制。在不同因素存在下 CRP 的交换行为表明，单体在转录复合物的结合和解离中具有重要意义。
