RESUMO
RNA-targeting small-molecule therapeutics is an emerging field hindered by an incomplete understanding of the basic principles governing RNA-ligand interactions. One way to advance our knowledge in this area is to study model systems where these interactions are better understood, such as riboswitches. Riboswitches bind a wide array of small molecules with high affinity and selectivity, providing a wealth of information on how RNA recognizes ligands through diverse structures. The cobalamin-sensing riboswitch is a particularly useful model system, as similar sequences show highly specialized binding preferences for different biological forms of cobalamin. This riboswitch is also widely dispersed across bacteria and therefore holds strong potential as an antibiotic target. Many synthetic cobalamin forms have been developed for various purposes including therapeutics, but their interaction with cobalamin riboswitches is yet to be explored. In this study, we characterize the interactions of 11 cobalamin derivatives with three representative cobalamin riboswitches using in vitro binding experiments (both chemical footprinting and a fluorescence-based assay) and a cell-based reporter assay. The derivatives show productive interactions with two of the three riboswitches, demonstrating simultaneous plasticity and selectivity within these RNAs. The observed plasticity is partially achieved through a novel structural rearrangement within the ligand binding pocket, providing insight into how similar RNA structures can be targeted. As the derivatives also show in vivo functionality, they serve as several potential lead compounds for further drug development.
Assuntos
Fenômenos Bioquímicos , Riboswitch , Vitamina B 12/metabolismo , Ligantes , RNA , Conformação de Ácido NucleicoRESUMO
Riboswitches are an outstanding example of genetic regulation mediated by RNA conformational switching. In these non-coding RNA elements, the occupancy status of a ligand-binding domain governs the mRNA's decision to form one of two mutually exclusive structures in the downstream expression platform. Temporal constraints upon the function of many riboswitches, requiring folding of complex architectures and conformational switching in a limited co-transcriptional timeframe, make them ideal model systems for studying these processes. In this review, we focus on the mechanism of ligand-directed conformational changes in one of the most widely distributed riboswitches in bacteria: the cobalamin family. We describe the architectural features of cobalamin riboswitches whose structures have been determined by x-ray crystallography, which suggest a direct physical role of cobalamin in effecting the regulatory switch. Next, we discuss a series of experimental approaches applied to several model cobalamin riboswitches that interrogate these structural models. As folding is central to riboswitch function, we consider the differences in folding landscapes experienced by RNAs that are produced in vitro and those that are allowed to fold co-transcriptionally. Finally, we highlight a set of studies that reveal the difficulties of studying cobalamin riboswitches outside the context of transcription and that co-transcriptional approaches are essential for developing a more accurate picture of their structure-function relationships in these switches. This understanding will be essential for future advancements in the use of small-molecule guided RNA switches in a range of applications such as biosensors, RNA imaging tools, and nucleic acid-based therapies.