THERMODYNAMIC LINKAGES IN THE CONTROL OF TRANSCRIPTION

大肠杆菌环腺苷酸受体蛋白（CRP）是关键的调节因子 负责细菌适应的来源的变化， 能源 CRP控制20多个基因的表达，这些基因编码 这些蛋白质将糖代谢为能量。 本实验室曾 开发了一个研究项目，定量地将各种反应联系起来， 参与CRP的调节活动， 个人的反应。 这些反应包括活化 CRP通过cAMP结合，cAMP诱导CRP结构改变， CRP-cAMP复合物与特异性DNA位点的结合，诱导的DNA 弯曲和RNA聚合酶与DNA-CRP-cAMP复合物的结合。 本研究的结果表明，CRP-cAMP的不对称性质 复合物可能是识别特定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复合物将利用其不对称性质使其自身定向， 非回文DNA半位点。 由于DNA结合位点 不同的操纵子呈现不同的方向， 不对称CRP-cAMP的DNA结合位点可能会导致 暴露于与RNA相互作用的CRP上的各种不同表面 聚合酶或其他转录因子。 这可能是 以区分CRP调控的20多个基因。 使用 一种CRP二聚体杂合体的组合， cAMP和DNA结合的不对称性， 将鉴定DNA-蛋白质复合物中的CRP。 而且 DNA半位点中序列差异的作用将通过 测定蛋白质-DNA结合常数和解离速率常数 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 还将监测CRP的构象变化。成果 调查应提供直接证据，以评估 CRP基因表达调控的不对称性。 以来 不对称CRP-cAMP复合物的形成是中心问题， 建议阐明CRP激活的机制， 通过定点突变产生的突变体。 突变位点 已经通过自然选择鉴定，这些突变体是 具有不同表型特征的单点突变。 这些站点位于CRP的不同部分。 因此，他们可能 干扰CRP激活途径的不同部分诱导 的cAMP。 用功能分析追踪结构扰动 可以为激活机制提供重要的见解。

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.