Structure and function analysis of a type III preQ(1)-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence

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(1) (preQ(1))-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ(1), 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(1)-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ(1) riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ(1) 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(1) riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ(1) over the chemically similar metabolic precursor preQ(0). Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ(1) riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.