Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

这项工作的目的是了解蛋白质对核酸的特异性识别的分子基础，以及在体内和体外它们调节相互作用的因素。这项拨款提案中描述的工作集中在大肠杆菌的Trp阻遏物(Trpr)上，目标如下：1)确定串联的序列要求，阻遏物与DNA的协同结合；2)确定体内阻遏物对自然操纵子的结合方式；3)在存在和不存在阻遏物的情况下，测量串联和单操纵子DNA的体外弯曲；4)与串联和单模式DNA结合相关的热力学参数的测量；5)结晶条件对特异和非特异DNA结合亲和力的影响的测定；以及6)TrpR片段的折叠和配体结合性质的测定。为了执行它们的调节功能，蛋白质对核酸序列进行精确的识别，在大量非特定序列的背景下正确地识别它们的真实靶标。在过去的四分之一个世纪里，生化和遗传学研究表明，特定复合体中的蛋白质和核酸伙伴之间存在高度的化学互补性。目前对Trpr-DNA相互作用的了解表明，特定和非特定结合之间的区别是由于各种因素的相互作用，包括微妙的能量考虑。这里提出的实验旨在为Trpr提供一个完整的图景。

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.