Mutational analysis of the purine riboswitch aptamer domain
Mutational analysis of the purine riboswitch aptamer domain
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DOI：
10.1021/bi700410g
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发表时间：
2007-11-20
期刊：
影响因子：
2.9
通讯作者：
Batey, Robert T.
中科院分区：
文献类型：
--
作者：
Gilbert, Sunny D.;Love, Crystal E.;Batey, Robert T.
嘌呤核糖开关是在5 '-非翻译区中常见的许多mRNA元件之一，其能够通过其直接结合小分子代谢物的能力以顺式方式控制表达。核糖开关的核碱基结合结构域（称为适体结构域）的广泛生物化学和结构分析表明，mRNA使用完全封装配体的复杂折叠的三向连接基序识别其同源配体。嘌呤核碱基的高亲和力结合通过腺嘌呤和鸟嘌呤核糖开关之间保守的远端环-环相互作用来促进。为了了解保守的核苷酸的贡献，在这两个三路交界处和环-环相互作用的RNA，我们进行了详细的诱变调查，这些元素的背景下的腺嘌呤响应的变体的xpt-pbuX鸟嘌呤核糖开关从枯草芽孢杆菌。通过等温滴定量热法测量的这些突变体结合配体的不同能力揭示了嘌呤结合所需的保守核苷酸的身份。五个突变体的结合形式的晶体学分析和其游离状态的化学探测表明，几个普遍保守的核苷酸的身份是不是必不可少的RNA-配体复合物的形成，而是保持一个结合能力的形式的游离RNA。这些数据表明，核糖开关中的保守模式是由配体结合复合物的形成、促进游离RNA的开放形式以及参与表达平台的二级结构开关的组合引起的。
The purine riboswitch is one of a number of mRNA elements commonly found in the 5'-untranslated region capable of controlling expression in a cis-fashion via its ability to directly bind small-molecule metabolites. Extensive biochemical and structural analysis of the nucleobase-binding domain of the riboswitch, referred to as the aptamer domain, has revealed that the mRNA recognizes its cognate ligand using an intricately folded three-way junction motif that completely encapsulates the ligand. High-affinity binding of the purine nucleobase is facilitated by a distal loop-loop interaction that is conserved between both the adenine and guanine riboswitches. To understand the contribution of conserved nucleotides in both the three-way junction and the loop-loop interaction of this RNA, we performed a detailed mutagenic survey of these elements in the context of an adenine-responsive variant of the xpt-pbuX guanine riboswitch from Bacillus subtilis. The varying ability of these mutants to bind ligand as measured by isothermal titration calorimetry uncovered the conserved nucleotides whose identity is required for purine binding. Crystallographic analysis of the bound form of five mutants and chemical probing of their free state demonstrate that the identity of several universally conserved nucleotides is not essential for formation of the RNA-ligand complex but rather for maintaining a binding-competent form of the free RNA. These data show that conservation patterns in riboswitches arise from a combination of formation of the ligand-bound complex, promoting an open form of the free RNA, and participating in the secondary structural switch with the expression platform.