THERMODYNAMIC LINKAGE IN THE CONTROL OF TRANSCRIPTION

大肠杆菌环磷酸腺苷受体(CRP)的正常功能模式需要 它需要在20多个特定的DNA序列和 细胞环境中存在的各种环核苷酸。在激活时 通过cAMP，CRP结合到特定的DNA位点。因此，C反应蛋白的活性模式 是多样性和特殊性之间的微妙平衡。然而，什么？ 结构元素通过调节 C反应蛋白-DNA接口？本实验室已鉴定出一些突变的CRP，其 它们对一系列DNA序列的亲和力排名不仅是 突变部位的功能也是特定身份的 结合的环核苷酸。因此，C反应蛋白是深入研究 阐明两者获得的调制机制 能量和结构信息。CAMP AS特异性的丧失 这些CRP突变体中的变构激活剂可能与 最近发现的DNA结合域中的额外cAMP结合位点 通过结晶学。因此，正在开发新的直接方法来产生 配基结合等温线，特别是与 确定配体占据的位置。调制机制可以 起源于C反应蛋白亚基或亚基间基性质的改变 沟通或两者兼而有之。因此，环核苷酸与环核苷酸的结合亲和力 将测定单体和二聚体的C反应蛋白。有间接的 当CRP假设蛋白质动力学发生变化的证据 各种功能状态。因此，突变对蛋白质的影响 动力学将通过使用FT-IR和MASS进行氢交换来监测 光谱分析。调制可以与非对称特性相关联 弯曲的女孩DNA。结构不对称在C反应蛋白-DNA相互作用中的作用 通过交联法和体外转录实验进行研究。这个 研究计划是对DNA的调制进行全面的生物物理研究 承认。

The normal functioning mode of E. coli cyclic AMP receptor (CRP) requires it to distinguish among the more than 20 specific DNA sequences and the various cyclic nucleotides present in the cellular milieu. Upon activation by cAMP, CRP binds to a specific DNA-site. Thus, the mode of CRP activity is a fine balance between diversity and specificity. However, what structural elements define diversity by modulating the interactions in the CRP-DNA interface? This laboratory has identified some mutant CRPs whose rank order of their affinity for a series of DNA sequences is not only a function of the site of mutation but also the specific identity of the bound cyclic nucleotide. Thus, CRP is prime for an in-depth study to elucidate the mechanism of modulation with the acquisition of both energetic and structural information. The loss of specificity for cAMP as an allosteric activator in these CRP mutants may be related to the additional cAMP binding site in the DNA binding domain identified recently by crystallography. Thus, new direct methods are being developed to yield ligand binding isotherms and, in particular, site-specific ones to identify the site occupied by the ligand. The mechanism of modulation may originate from a change in properties of the CRP subunit or inter-subunit communication or both. Thus, the binding affinity of cyclic nucleotides to both monomeric and dimeric CRP will be determined. There is indirect evidence to indicate a change in protein dynamics as CRP assumes the various functional states. Hence, the effect of mutation on protein dynamics will be monitored by hydrogen exchange using FT-IR and mass spectrometry. Modulation may be linked to the asymmetric nature of the bent gal DNA. The role of structural asymmetry in CRP-DNA interaction will be investigated by cross linking and in vitro transcription assays. The research program is a comprehensive biophysical study on modulation in DNA recognition.