Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Nucleic Acids Res. 2023 Mar 21;51(5):2464-2484. doi: 10.1093/nar/gkad056.
Riboswitches regulate downstream gene expression by binding cellular metabolites. Regulation of translation initiation by riboswitches is posited to occur by metabolite-mediated sequestration of the Shine-Dalgarno sequence (SDS), causing bypass by the ribosome. Recently, we solved a co-crystal structure of a prequeuosine1-sensing riboswitch from Carnobacterium antarcticum that binds two metabolites in a single pocket. The structure revealed that the second nucleotide within the gene-regulatory SDS, G34, engages in a crystal contact, obscuring the molecular basis of gene regulation. Here, we report a co-crystal structure wherein C10 pairs with G34. However, molecular dynamics simulations reveal quick dissolution of the pair, which fails to reform. Functional and chemical probing assays inside live bacterial cells corroborate the dispensability of the C10-G34 pair in gene regulation, leading to the hypothesis that the compact pseudoknot fold is sufficient for translation attenuation. Remarkably, the C. antarcticum aptamer retained significant gene-regulatory activity when uncoupled from the SDS using unstructured spacers up to 10 nucleotides away from the riboswitch-akin to steric-blocking employed by sRNAs. Accordingly, our work reveals that the RNA fold regulates translation without SDS sequestration, expanding known riboswitch-mediated gene-regulatory mechanisms. The results infer that riboswitches exist wherein the SDS is not embedded inside a stable fold.
Riboswitches 通过结合细胞代谢物来调节下游基因表达。假定通过代谢物介导的 Shine-Dalgarno 序列（SDS）隔离来调节翻译起始，从而导致核糖体旁路。最近，我们解决了南极光杆菌（Carnobacterium antarcticum）中一个前假尿嘧啶核苷 1 感应核糖开关的共晶结构，该核糖开关在单个口袋中结合两种代谢物。该结构表明，基因调控 SDS 内的第二个核苷酸 G34 参与晶体接触，掩盖了基因调控的分子基础。在这里，我们报告了一个共晶结构，其中 C10 与 G34 配对。然而，分子动力学模拟显示该配对迅速溶解，无法重新形成。活细菌细胞内的功能和化学探测实验证实了 C10-G34 配对在基因调控中的非必要性，从而提出了紧凑的假结折叠足以进行翻译衰减的假设。值得注意的是，当使用长达 10 个核苷酸的无结构间隔物将南极光杆菌适体与 SDS 分离时，它仍然保留了显著的基因调控活性——类似于 sRNA 采用的空间位阻。因此，我们的工作表明，RNA 折叠可以在不隔离 SDS 的情况下调节翻译，扩展了已知的核糖开关介导的基因调控机制。结果推断出，某些核糖开关中 SDS 并未嵌入稳定的折叠中。
