Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
J Biol Chem. 2023 Oct;299(10):105208. doi: 10.1016/j.jbc.2023.105208. Epub 2023 Sep 1.
Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine (preQ)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ over the chemically similar metabolic precursor preQ. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.
Riboswitches 是一类主要存在于细菌信使 RNA 5' 前导区的小非编码 RNA，可通过结合一种或多种细胞代谢物来调节下游基因的表达。这种非编码 RNA 通常在翻译水平上受到调控，这被认为是由 Shine-Dalgarno 序列（SDS）核糖体结合位点的可及性介导的。目前已知有三类（I-III）前 queuosine（preQ）感应 riboswitch 控制翻译。I 类又分为三个亚型（I-III），它们具有不同的感应 preQ 的机制，preQ 参与 queuosine 生物合成。为了深入了解翻译调控，我们通过生物信息学搜索，确定了一种在大肠杆菌（Eco）中发现的 I 类 III 型 preQ 感应 riboswitch 的 2.30 Å 分辨率共晶结构。Eco riboswitch 的结构与之前的 preQ riboswitch 结构不同，因为它具有最小的天然存在的适体，并且 SDS 直接与 preQ 代谢物接触。我们使用表面等离子体共振和体内基因表达测定验证了结构观察结果，结果表明在活的 E. coli 中具有强烈的开关作用。我们的结果表明，Eco riboswitch 对破坏形成假结的非canonical 相互作用的突变相对敏感。与 II 类 preQ riboswitch 不同，动力学分析表明 III 型 Eco riboswitch 强烈偏爱 preQ 而不是化学上相似的代谢前体 preQ。我们的结果揭示了非canonical 相互作用在 riboswitch 驱动的基因调控中的重要性，以及 I 类 preQ riboswitch 假结作为支持 SDS 隔离的代谢物感应平台的多功能性。
