A Modular RNA Domain That Confers Differential Ligand Specificity

Abstract

The modularity of protein domains is well-known, but the existence of independent domains that confer function in RNA is less established. Recently, a family of RNA aptamers termed ykkC was discovered; they bind at least four ligands of very different chemical composition, including guanidine, phosphoribosyl pyrophosphate (PRPP), and guanosine tetraphosphate (ppGpp) (graphical abstract). Structures of these aptamers revealed an architecture characterized by two coaxial helical stacks. The first helix appears to be a generic scaffold, while the second helix forms the most contacts to the ligands. To determine if these two regions within the aptamer are modular units for ligand recognition, we swapped the ligand-binding coaxial stacks of a guanidine aptamer and a PRPP aptamer. This operation, in combination with a single mutation in the scaffold domain, achieved full switching of ligand specificity. This finding suggests that the ligand-binding helix largely dictates the ligand specificity of ykkC RNAs and that the scaffold coaxial stack is generally compatible with various ykkC ligand-binding modules. This work presents an example of RNA domain modularity comparable to that of a ligand-binding protein, showcasing the versatility of RNA as an entity capable of molecular evolution through adaptation of existing motifs.

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2′-deoxyguanosine

“…Given RNA’s limited chemical and structural diversity, it is not surprising that other riboswitch aptamer architectures also have diversified to selectively sense more than one purine-containing ligand. For example, variants of c-di-GMP-I riboswitches are known that sense c-AMP-GMP ( 33 , 34 ), and riboswitches that sense ppGpp ( 35 , 36 ) or ADP ( 20 ) are based on the same ykkC motif scaffold ( 41 ). Exploiting variants of a few aptamer scaffolds to diversify the types of purines that can be sensed would reduce the evolutionary burden required to produce novel aptamer architectures for each new molecular target.…”

“…Thus, when mutations occur at otherwise highly conserved nucleotide positions within the binding pockets of aptamers, it is likely that the variant RNAs have adapted to change their ligand-binding specificity ( 25 ). Examples of such evolutionary changes have been reported for several riboswitch classes, including guanine riboswitch variants that recognize adenine ( 26 ) or 2′-deoxyguanosine ( 25 , 27 ), adenosylcobalamin riboswitch variants that sense aquocobalamin ( 28 , 29 ), FMN riboswitch variants that sense chemical derivatives of this enzyme cofactor ( 25 , 30 – 32 ), c-di-GMP riboswitch variants that sense c-AMP-GMP ( 33 , 34 ), and guanidine-I riboswitch variants that sense ppGpp ( 35 , 36 ), PRPP ( 36 , 37 ), or ADP ( 20 ). Numerous additional changes in ligand specificities for variant riboswitch aptamers have also been proposed ( 17 , 20 , 25 ).…”

Identification and characterization of RNA binding sites for (p)ppGpp using RNA-DRaCALA

“…It is possible that an RNA aptamer could have evolved to prefer one conformation of (p)ppGpp or the other. Additional mutational and biochemical analysis and crystal structures of ligand bound aptamers, such as those performed to compare PRPP- and ppGpp-binding ( 44 , 47 ), or PRPP- and guanidine binding ( 51 ), will be required to further identify sequence and structure determinants important for ligand binding and the specificity for similar but distinct ligands such as ppGpp and pppGpp.…”

Siblings or doppelgängers? Deciphering the evolution of structured cis-regulatory RNAs beyond homology

“…A single G96A point mutation switches the ligand affinity for the PRPP-binding aptamer to ppGpp with a 40 000-fold change in selectivity ( Figure 2B) [11]. A single mutation to the structural core along with swapping the ligand-binding helix converts the PRPP aptamer to a guanidine aptamer [13]. The selective pressures driving the evolution of the guanidine, ppGpp, and PRPP-sensing ykkC riboswitch classes thus show how subtle sequence changes and modularity can lead to the adaptability of RNA-based regulation from a shared core structure.…”