Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

这项工作的目的是了解分子基础， 蛋白质对核酸的特异性识别， 参与它们的调节相互作用，无论是在体内， 体外 本拨款申请中描述的工作重点是 trp阻遏蛋白。大肠杆菌（TrpR），具有以下目标：1） 串联、合作和运输的顺序要求的确定 阻遏物与DNA的结合; 2）结合模式的确定 在自然操纵子上由阻遏物在体内使用; 3）测量 串联和单操纵基因DNA在体外的弯曲， 阻遏物的存在和不存在; 4）测量 与串联DNA结合相关的热力学参数 和单模; 5）结晶效果的测定 特异性和非特异性DNA结合亲和力的条件;以及6） 确定一个具有折叠和配体结合特性的 TrpR片段。 %%% 为了执行它们的调节功能，蛋白质精确地 识别核酸序列，正确识别其 真正的目标对非特异性序列的巨大背景。 在过去的四分之一世纪里，生物化学和遗传学研究 指出了化学物质之间的高度互补性， 在特定复合物中的蛋白质和核酸伴侣。 的 目前关于TrpR-DNA相互作用的知识表明， 特异性结合和非特异性结合的区别在于 由于各种因素的相互作用，包括微妙的能量 注意事项。 这里提出的实验旨在 为TrpR提供了一个完整的图像。

This work is aimed at understanding the molecular basis for specific recognition of nucleic acids by proteins, and the factors involved in their regulatory interactions, both in vivo and in vitro. The work described in this grant proposal focuses on the trp repressor of E. coli (TrpR), with the following goals: 1) Determination of the sequence requirements for tandem, cooperative binding of repressor to DNA; 2) Determination of the binding mode used by the repressor in vivo on natural operators; 3) Measurement of the bending of tandem and single operator DNA's in vitro in the presence and absence of repressor; 4) Measurement of the thermodynamic parameters associated with DNA binding in the tandem and single modes; 5) Determination of the effect of crystallization conditions on specific and nonspecific DNA binding affinity; and 6) Determination of the folding and ligand-binding properties of a fragment of TrpR. %%% To carry out their regulatory functions, proteins exercise precise recognition of nucleic acid sequences, correctly identifying their true targets against a vast background of nonspecific sequences. In the last quarter century, biochemical and genetic studies have pointed to a high degree of chemical complementarily between the protein and nucleic acid partners in specific complexes. The current state of knowledge about the TrpR-DNA interaction suggests that the distinction between specific and non specific binding is due to an interplay of factors, including subtle energetic considerations. The experiments proposed here are aimed at provided a complete picture for TrpR.