Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

这项工作旨在了解蛋白质特异性识别核酸的分子基础，以及它们在体内和体外的调节相互作用中涉及的因素。本项目主要研究大肠杆菌的trp抑制因子（TrpR），目标如下：1)确定抑制因子与DNA串联、协同结合的序列要求；2)确定抑制物在体内对天然载体的结合方式；3)在体外测定抑制因子存在和不存在时串联和单操作子DNA的弯曲；4)串联和单模式DNA结合的热力学参数测量；5)结晶条件对特异性和非特异性DNA结合亲和力影响的测定；6)确定TrpR片段的折叠和配体结合特性。为了实现它们的调节功能，蛋白质对核酸序列进行精确的识别，在大量的非特异性序列背景下正确地识别出它们的真正目标。在过去的25年里，生物化学和遗传学的研究已经指出，在特定的复合物中，蛋白质和核酸伙伴之间存在高度的化学互补。目前关于TrpR-DNA相互作用的知识表明，特异性和非特异性结合之间的区别是由于各种因素的相互作用，包括微妙的能量因素。本文提出的实验旨在为TrpR提供一个完整的图像。

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.