THERMODYNAMIC LINKAGE IN THE CONTROL OF TRANSCRIPTION

E.大肠杆菌环腺苷酸受体（CRP）需要 它可以区分20多个特定的DNA序列， 存在于细胞环境中的各种环核苷酸。在激活时 通过cAMP，CRP与特定的DNA位点结合。因此，CRP活性的模式 是多样性和特殊性之间的一种微妙平衡。不过 结构要素通过调节 CRP-DNA接口这个实验室已经发现了一些突变的CRP， 它们对一系列DNA序列的亲和力的等级顺序不仅是一个 突变位点的功能，而且突变位点的特异性身份， 结合的环核苷酸。因此，CRP是进行深入研究的首要条件， 阐明调制的机制与收购的两个 能量和结构信息。cAMP特异性的丧失， 这些CRP突变体中的变构激活剂可能与 最近在DNA结合结构域中发现了一个额外的cAMP结合位点 通过结晶学因此，正在开发新的直接方法以产生 配体结合等温线，特别是位点特异性等温线， 识别配体占据的位点。调制机制可能 来源于CRP亚基或亚基间性质的改变 沟通或两者。因此，环核苷酸与 将测定单体和二聚体CRP。有间接 有证据表明CRP假设蛋白质动力学发生变化， 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和质谱的氢交换来监测。 光谱法调制可能与非对称性质有关， 弯曲的半乳糖DNA结构不对称在CRP-DNA相互作用中的作用将 通过交联和体外转录测定进行研究。的 研究计划是一个全面的生物物理学研究的调制DNA 识别.

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.