Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch

The cyclic diguanylate [bis-(3'–5')-cyclic dimeric guanosine monophosphate, c-di-GMP] riboswitch is the first known example of a gene-regulatory RNA that binds to a second messenger. C-di-GMP is widely employed by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The 2-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch employing canonical and non-canonical base pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.