Yang J, Gunasekera A, Lavoie T A, Jin L, Lewis D E, Carey J
Chemistry Department Princeton University, NJ 08544-1009, USA.
J Mol Biol. 1996 Apr 26;258(1):37-52. doi: 10.1006/jmbi.1996.0232.
The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively using in vitro and in vivo methods. DNA sequence requirements for 1:1 and tandem 2:1 (TrpR:DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is 3'CNTGA5'5'GNACT3', consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2:1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2:1 complex, or with an 8-base-pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprinting in vitro of 1:1 and 2:1 complexes formed sequentially on the two unequal half-site pairs of the trpEDCBA operator from Serratia marcescens indicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting of Escherichia coli operators in vivo showed that, over a wide range of intracellular TrpR concentration, the trpEDCBA operator is occupied by three repressor dimers, aroH is occupied by two dimers, and the 1:1 binding mode is used on the trpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of approximately -2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varies with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation.
利用体外和体内方法对色氨酸阻遏物（TrpR）与其操纵基因的结合进行了定量研究。研究了在各种序列背景下1:1和串联2:1（TrpR:DNA）结合的DNA序列要求。结果表明，一个TrpR二聚体的一个螺旋-转角-螺旋基序识别的最佳半位点序列是3'CNTGA5'5'GNACT3'，这与1:1和2:1共结晶复合物的X射线衍射分析观察到的接触情况一致。半位点可以通过直接邻接配对形成回文结构，形成串联2:1复合物的成核位点，或者通过8个碱基对的间隔形成1:1靶标。在粘质沙雷氏菌trpEDCBA操纵基因的两个不相等半位点对上依次形成的1:1和2:1复合物的体外硫酸二甲酯（DMS）甲基化保护足迹表明，位点占据存在严格的层次结构，其中一个半位点对作为串联结合的成核位点。体内大肠杆菌操纵基因的DMS足迹表明，在广泛的细胞内TrpR浓度范围内，trpEDCBA操纵基因被三个阻遏物二聚体占据，aroH被两个二聚体占据，并且在trpR操纵基因上使用1:1结合模式。这些不同占据状态的共存意味着蛋白质浓度的变化仅影响每个操纵基因的占据分数而不是结合模式，结合模式由操纵基因区域中存在的半位点序列数量决定。使用含有与天然操纵基因中相同的最佳位点且间隔相同的对称合成操纵基因通过凝胶阻滞法测量的串联复合物形成的协同性适中，这意味着最大耦合自由能约为-2千卡/摩尔。在其他序列上，协同性的表观程度以及表观亲和力随序列和序列背景而变化，其方式与结构模型一致，这表明亲和力和协同性之间的补偿是一种允许操纵基因序列变异耐受的机制。
