THERMODYNAMIC LINKAGES IN THE CONTROL OF TRANSCRIPTION

大肠杆菌cAMP受体蛋白(Crp)是关键的调节因子 负责细菌适应源头的变化 能量。C反应蛋白控制20多个基因的表达，这些基因编码 将糖代谢为能量的蛋白质。这个实验室有 开发了一项研究计划，将各种反应定量地联系起来 通过监测许多参与CRP调节活动的基因 每个人的反应。这些反应包括激活 通过cAMP结合，cAMP诱导的CRP的结构变化， CRP-cAMP复合体与特定DNA位点的结合，诱导的DNA RNA聚合酶与DNA-CRP-cAMP复合体的弯曲和结合。 这项研究的结果表明，CRP-cAMP的不对称性质 复合体可能是识别特定DNA的重要特征 不同操纵子中的结合位点。有人提出，不对称 CRP-cAMP复合体将利用其不对称的性质来定位自己 非回文DNA的一半位置。由于DNA结合位点在 不同的操纵子呈现不同的方向，一个 不对称的CRP-cAMP到DNA结合部位可能会导致 暴露于C反应蛋白表面与RNA相互作用的不同表面 聚合酶或其他转录因子。这可能是一种机制 以便于CRP区分由CRP调控的20多个基因。vbl.使用 CRP二聚体的杂交体组合被专门设计成 在cAMP和DNA结合上不对称， DNA-蛋白质复合体中的CRP将被鉴定出来。此外， 序列差异在DNA半位点中的作用将通过 蛋白质-DNA结合常数和解离速率常数的测定 除了对DNA-CRP-RNA形成的亲和力之外 聚合酶复合体。DNA半位点诱导的能力 还将监测CRP的构象变化。这样做的结果是 调查应提供直接证据，以评估 C反应蛋白基因表达调控的不对称性。自.以来 不对称的CRP-cAMP复合体的形成是中心问题，它是 提出了在阐明C反应蛋白激活机制的基础上 通过定点突变产生的突变体。突变的部位 是通过自然选择来鉴定的，这些突变体是产物 具有不同表型特征的单点突变。 这些部位位于C反应蛋白的不同部位。因此，他们可能会 干预C反应蛋白活化途径的不同环节 通过露营。用功能分析追踪结构扰动 可以为激活机制提供重要的洞察力。

E.coli cyclic AMP receptor protein (CRP) is the key regulatory factor responsible for the bacteria to adapt to a change in the source of energy. CRP controls the expression of more than 20 genes which code the proteins that metabolize the sugars for energy. This laboratory has developed a research program to quantitatively link the various reactions that are involved in the regulatory activity of CRP by monitoring many of the individual reactions. These reactions include the activation of CRP by cAMP binding, the structural changes in CRP induced by cAMP, the binding of CRP-cAMP complex to the specific DNA site, the induced DNA bending and the binding of RNA polymerase to the DNA-CRP-cAMP complex. Results of this study indicate that the asymmetric nature of the CRP-cAMP complex may be an important feature in recognizing the specific DNA binding site in different operons. It is proposed that the asymmetric CRP-cAMP complex will utilize its asymmetric nature to orient itself with the non-palindromic DNA half sites. Since the DNA binding sites in different operons assume different orientations, the binding of an asymmetric CRP-cAMP to the DNA binding site can potentially lead to a variety of different surfaces on CRP exposed for interaction with RNA polymerase or other transcription factors. This might be the mechanism for CRP to distinguish the more than 20 genes regulated by CRP. Using a combination of hybrids of CRP dimer specifically engineered to be asymmetric in cAMP and DNA binding, the specific orientation adopted by CRP in the DNA-protein complex will be identified. Furthermore, the roles of the sequence difference in the DNA half-sites will be probed by measuring the protein-DNA binding constant and dissociation rate constant in addition to the affinity for the formation of the DNA-CRP-RNA polymerase complex. The ability of the DNA half-sites to induce conformational changes in CRP will also be monitored. Results of this investigation should provide direct evidence to assess the role of asymmetry in the regulation of gene expression of CRP. Since the formation of the asymmetric CRP-cAMP complex is the central issue, it is proposed to elucidate the mechanism of activation of CRP with the help of mutants generated by site-directed mutagenesis. The sites of mutation have been identified by natural selections and these mutants are products of single site mutations with different phenotypic characteristics. These sites are located at different parts of CRP. Thus, they might interfere with different parts of the pathway of CRP activation induced by cAMP. Tracking the structural perturbation with functional assays could provide significant insight to the mechanism of activation.