Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain
Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain
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DOI：
10.1016/j.jmb.2006.04.003
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发表时间：
2006-06-09
影响因子：
5.6
通讯作者：
Batey, Robert T.
中科院分区：
文献类型：
--
作者：
Gilbert, Sunny D.;Stoddard, Colby D.;Batey, Robert T.
核糖开关是常见于细菌mRNA中的顺式作用遗传调节元件，其由与调节开关偶联的代谢物响应性适体结构域组成。嘌呤核糖开关响应细胞内腺嘌呤或鸟嘌呤/次黄嘌呤的浓度来控制基因表达。嘌呤核糖开关的适体结构域含有嘧啶残基（Y 74），其与结合的嘌呤核碱基配体形成沃森-克里克碱基配对相互作用，其区分腺嘌呤和鸟嘌呤。我们试图了解这种特异性的结构基础和嘌呤核糖开关识别配体的机制。在这里，我们提出了2，6-二氨基嘌呤结合结构的C74 U突变的xpt-pbuX鸟嘌呤核糖开关，沿着与详细的热力学和动力学分析的核碱基识别的天然和突变的核糖开关。这些研究清楚地表明，在位置74的嘧啶是嘌呤核糖开关特异性的唯一决定因素。此外，突变型核糖开关结合腺嘌呤和腺嘌呤衍生物相比，鸟嘌呤响应核糖开关。在我们的实验条件下，2，6-二氨基嘌呤与RNA结合的Δ H = -40.3 kcal mol（-1），Δ S = -97.6 cal mol（-1）K-1，Δ G = -10.73 kcal mol（-1）。在不同的实验条件下，对缓慢的结合速率（2-氨基嘌呤结合腺嘌呤响应突变型核糖开关和7-脱氮鸟嘌呤结合鸟嘌呤核糖开关分别为0.15 × 10（5）M-1 s（-1）和2.1 × 10（5）mM（-1）s（-1））进行动力学测定，使我们能够提出嘌呤核糖开关识别配体的机制。结合口袋的构象动态未配体状态首先通过配体和Y 74之间的Watson-Crick碱基配对以及随后的f2/3环的排序来稳定，从而将配体封闭在三通接头内。(c)2006爱思唯尔有限公司保留所有权利。
Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diamino-purine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with Delta H = -40.3 kcal mol(-1), Delta S = -97.6 cal mol(-1) K-1, and Delta G = -10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5) M-1 s(-1) and 2.1 x 10(5) mM(-1) s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the f2/3 loop, enclosing the ligand within the three-way junction. (c) 2006 Elsevier Ltd. All rights reserved.