Abstract
Riboswitches are metabolite-sensing, conserved domains located in non-coding regions of mRNA that are central to regulation of gene expression. Here we report the first three-dimensional structure of the recently discovered S-adenosyl-L-methionine responsive SAM-VI riboswitch. SAM-VI adopts a unique fold and ligand pocket that are distinct from all other known SAM riboswitch classes. The ligand binds to the junctional region with its adenine tightly intercalated and Hoogsteen base-paired. Furthermore, we reveal the ligand discrimination mode of SAM-VI by additional X-ray structures of this riboswitch bound to S-adenosyl-L-homocysteine and a synthetic ligand mimic, in combination with isothermal titration calorimetry and fluorescence spectroscopy to explore binding thermodynamics and kinetics. The structure is further evaluated by analysis of ligand binding to SAM-VI mutants. It thus provides a thorough basis for developing synthetic SAM cofactors for applications in chemical and synthetic RNA biology.
Highlights
Riboswitches are metabolite-sensing, conserved domains located in non-coding regions of mRNA that are central to regulation of gene expression
The secondary structure model for the SAM-VI riboswitch predicts the formation of three stems P1, P2, and P3 that are connected by one central 3-way junction and that do not contribute to formation of any obvious long-range interactions (Supplementary Fig. 1b)[13]
One transcript from B. angulatum 59 metK in which the U1A recognition site had been introduced as terminal loop of stem P2 yielded diffraction quality crystals when co-crystallized with the U1A protein (Fig. 1a)
Summary
Riboswitches are metabolite-sensing, conserved domains located in non-coding regions of mRNA that are central to regulation of gene expression. 1234567890():,; Riboswitches are gene regulatory elements commonly located in the 5’-untranslated regions (5′-UTRs) of bacterial mRNAs1,2 They consist of two functional domains, the ligand-sensing aptamer and the downstream adjoining expression platform. In particular the SAM-riboswitch families provide a perfect setting for investigations on how the same cognate ligand can be recognized by different RNA architectures Towards this end, X-ray crystallographic and NMR-spectroscopic structural research has made significant contributions[25,26,27,28,29,30] to shed light on the distinct ligand recognition modes, which provide the basis for the cellular function of SAM riboswitches. The conserved sequence and secondary structure of SAM-VI (Supplementary Fig. 1b) has vague similarities with the SAM-III riboswitch Both SAM-III and SAM-VI consist of three stems (P1, P2, and P3) that are connected by one central 3-way junction, and both riboswitches selectively bind SAM over. Many more nucleotides are uniquely conserved in the consensus sequence of SAM-VI compared to SAM-III13
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