Ligand specificity is an essential requirement for all riboswitches. Some variant riboswitches utilize a common structural motif, yet through subtle sequence differences, they are able to selectively respond to different small molecule ligands and regulate downstream gene expression. These variants discriminate between structurally and chemically similar ligands. Crystal structures provide insight into how specificity is achieved. However, ligand specificity cannot always be explained solely by nucleotides in direct contact with the ligand. The cyclic dinucleotide variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP riboswitches, that were distinguished based on the identity of a single nucleotide in contact with the ligand. Here we report a variant riboswitch with a mutation at a second ligand-contacting position that is promiscuous for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT, was used to quantitatively assess thousands of sites in the first- and second-shells of ligand contact for impacts on ligand specificity and promiscuity. In addition to nucleotides in direct ligand contact, nucleotides more distal from the binding site, within the J1/2 linker and the terminator helix, were identified that impact ligand specificity. These findings provide an example of how nucleotides outside the ligand binding pocket influence the riboswitch specificity. Moreover, these distal nucleotides could be used to predict promiscuous sequences.

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第二壳核苷酸对环状二核苷酸核糖开关配体特异性的影响

配体特异性是所有核糖开关的基本要求。一些变异核糖开关利用共同的结构基序，但通过细微的序列差异，它们能够选择性地响应不同的小分子配体并调节下游基因表达。这些变体区分结构和化学上相似的配体。晶体结构提供了对如何实现特异性的深入了解。然而，配体特异性不能总是仅通过与配体直接接触的核苷酸来解释。环状二核苷酸变体家族包含两类：环状二 GMP 和环状 AMP-GMP 核糖开关，它们是根据与配体接触的单个核苷酸的身份来区分的。在这里，我们报告了一种在第二个配体接触位置处具有突变的核糖开关变体，尽管预计会优先选择环-AMP-GMP，但该突变对于环-二-GMP和环-AMP-GMP都是混杂的。高通量突变分析 SMARTT 用于定量评估配体接触的第一和第二壳中的数千个位点对配体特异性和混杂性的影响。除了与配体直接接触的核苷酸外，J1/2 连接子和终止子螺旋内距离结合位点更远的核苷酸也被确定会影响配体特异性。这些发现提供了配体结合袋外的核苷酸如何影响核糖开关特异性的例子。此外，这些远端核苷酸可用于预测混杂序列。