A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition

Abstract

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.

Thoughts on how to think (and talk) about RNA structure

“…For other RNAs, the landscape could have a few deep wells that represent more than one favored distinct conformation, which can interconvert. In some cases, these conformations could represent one or more “misfolded” states or stable intermediates ( 31 – 33 ). Some RNA sequences may instead have flatter folding landscapes, or flatter regions within larger landscapes, with several shallow indentations.…”

Potential effects of metal ion induced two-state allostery on the regulatory mechanism of add adenine riboswitch

“…However, they are of great value in refining the understanding of regulation mechanisms of riboswitches, and experiments are therefore dedicated to their exploration with improved techniques. Although the structural changes in purine riboswitch and some of the important intermediate states that accompany this process have been studied experimentally to some extent, the specific roles of metal ions in these issues are still poorly understood, mainly due to the following limitations of current experimental techniques: (i) experiments based on X-ray diffraction only yield transient structures of individual intermediate states of the riboswitches, but cannot describe dynamic details of the conformational transition processes involved; (ii) while NMR and single-molecule FRET can be used to study the dynamics of biomolecules in solution and indirectly speculate on the effect of metal ions on them 36 , 39 , neither can give direct evidence of the roles of metal ions in the successive conformational transitions that occur in riboswitches 40 ; (iii) even in X-ray crystallography, it is quite difficult to identify Mg 2+ ions (most effective for stabilizing the native RNA structure) by checking residual electron density maps because they have the same number of electrons as Na + ions and water molecules 41 ; (iv) the contribution of monovalent metal ions (such as K + and Na + ) in the free or weakly bound state to the stability of the RNA structure cannot be ignored either 12 , 42 , but almost all experimental techniques are unable to assess it in detail.

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A transient conformation facilitates ligand binding to the adenine riboswitch

“…In general, aptamer domains with available crystal structures have been studied widely. For example, the aptamer of the adenine riboswitch, which is ∼70 nucleotides (nt), has been characterized by various methods ( Broft et al., 2020 ; Dalgarno et al., 2013 ; Greenleaf et al., 2008 ; Neupane et al., 2011 ; Noeske et al., 2005 ; Serganov et al., 2004 ; St-Pierre et al, 2021 ), and Wang group captured a holo (ligand-bound), two apo (ligand-free), and an intermediate conformation with a re-arranged binding pocket and identified the stability of P1 was strengthened after ligand binding by mix-and-inject XFEL serial crystallography ( Stagno et al., 2017 ). However, the full-length adenine riboswitch (termed the adenine riboswitch in subsequent text), which is ∼120 nt in length, is poorly understood, and only a few studies have examined the dynamic switch of the full-length riboswitch ( Frieda and Block, 2012 ; Reining et al., 2013 ; Warhaut et al., 2017 ; Tomezsko et al., 2020 ; Tian et al., 2018 ).…”