Ligand-induced folding of the guanine-sensing riboswitch is controlled by a combined predetermined induced fit mechanism (original) (raw)
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Nucleic Acids Research, 2006
Riboswitches are highly structured elements in the 5 0 -untranslated regions (5 0 -UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/ hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by longrange base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using highresolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg 2+ -ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg 2+ -dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.
Nucleic Acids Research, 2010
Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated preformation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg 2+ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gsw loop ) of the guanine-sensing riboswitch and their Mg 2+induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gsw loop or the increase in temperature for both Gsw and Gsw loop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg 2+ . We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.
Biochemistry, 2012
In this study, we employed a combination of steady-state and time-resolved fluorescence spectroscopy and studied the site-specific dynamics in a GTP aptamer using 2-aminopurine as a fluorescent probe. We compared the dynamics of the GTP-bound aptamer with that of the free aptamer as well as when it is denatured. GTP binding leads to an overall compaction of structure in the aptamer. The general pattern of fluorescence lifetimes and correlation times scanned across several locations in the aptamer does not seem to change following GTP binding. However, a remarkable narrowing of the lifetime distribution of the aptamer ensues following its compaction by GTP binding. Interestingly, such a "conformational narrowing" is evident from the lifetime readouts of the nucleotide belonging to the stem as well as the "bulge" part of the aptamer, independent of whether it is directly interacting with GTP. Taken together, these results underscore the importance of an overall intrinsic structure associated with the free aptamer that is further modulated following GTP binding. This work provides strong support for the "conformational selection" hypothesis of ligand binding. RNA aptamers are RNA oligonucleotides that can bind specifically to a wide range of target molecules such as small molecules, cofactors, amino acids, etc., with high affinity. 1−3 Aptamers are selected from a combinatorial library through an artificial evolution procedure called systematic evolution of ligands by exponential enrichment (SELEX). 4,5 There has been an exponentially growing number of applications of aptamers, namely, as biosensors, 6,7 as a tool for metabolite sensing, 8 as diagnostics or biomedical applications, 9 and as drug delivery tools. 10 One of the main attractive properties of aptamers is the in vitro selection procedure that can provide an aptamer against virtually any target of choice and with a predefined affinity. The structural flexibility of oligonucleotides allows for adaptation of the aptamer to various structures. 1,11−20 The recognition and binding of a small molecule ligand by nucleic acids are thought to be based mainly on stacking interactions and hydrogen bonding. 18,21,22 These types of interactions are crucial for RNA function in nature as well as in the context of RNA as a drug target. 23,24 RNA aptamers are ideal systems for detailed studies of these interactions. 25 Several structures of aptamer (both artificial and natural)−ligand complexes have been determined using X-ray crystallography 26−30 and nuclear magnetic resonance (NMR) spectroscopy. 14−23,31−37 The structures of complexes of aptamers with ATP, biotin, or FMN, among others, demonstrated that specificity is most often achieved by specific hydrogen bonding patterns and stacking of aromatic ring systems in the ligand with bases in the RNA. 21,31,38 Another characteristic feature of these complexes, especially those with artificially selected aptamers, is the fact that the RNA binding pocket in its ligand free form is largely unstructured and folding occurs simultaneously with ligand binding, a process that has been termed conformational selection or adaptive binding. 19,22,39,40 Artificially selected RNA aptamers are quite small in size, very similar to the aptamer domain of RNA switches. 28−37,41,42 In RNA switches, structural changes in the aptamer domain caused by ligand binding led to the switching action. Understanding the intricacies of ligand-induced changes in
An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters
Physical chemistry chemical physics : PCCP, 2017
Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor's dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state. In this article, we question whether a polarized division of RNA binding mechanisms truly meets the conceptual needs of the field. We will review the history behind this classification of RNA-ligand interactions, and the way induced fit in particular has been rehabilitated by single-molecule studies of RNA aptamers. We will highlight several recent results from single-molecule experimental studies of riboswitches that reveal gaps o...
RNA, 2009
Riboswitches are structural cis-acting genetic regulatory elements in 59 UTRs of mRNAs, consisting of an aptamer domain that regulates the behavior of an expression platform in response to its recognition of, and binding to, specific ligands. While our understanding of the ligand-bound structure of the aptamer domain of the adenine riboswitches is based on crystal structure data and is well characterized, understanding of the structure and dynamics of the ligand-free aptamer is limited to indirect inferences from physicochemical probing experiments. Here we report the results of 15-nsec-long explicit-solvent molecular dynamics simulations of the add A-riboswitch crystal structure (1Y26), both in the adenine-bound (CLOSED) state and in the adenine-free (OPEN) state. Root-mean-square deviation, root-mean-square fluctuation, dynamic cross-correlation, and backbone torsion angle analyses are carried out on the two trajectories. These, along with solvent accessible surface area analysis of the two average structures, are benchmarked against available experimental data and are shown to constitute the basis for obtaining reliable insights into the molecular level details of the binding and switching mechanism. Our analysis reveals the interaction network responsible for, and conformational changes associated with, the communication between the binding pocket and the expression platform. It further highlights the significance of a, hitherto unreported, noncanonical W:H trans base pairing between A73 and A24, in the OPEN state, and also helps us to propose a possibly crucial role of U51 in the context of ligand binding and ligand discrimination.
Molecules, 2019
Aptamer selection can yield many oligonucleotides with different sequences and affinities for the target molecule. Here, we have combined computational and experimental approaches to understand if aptamers with different sequences but the same molecular target share structural and dynamical features. NEO1A, with a known NMR-solved structure, displays a flexible loop that interacts differently with individual aminoglycosides, its ligand affinities and specificities are responsive to ionic strength, and it possesses an adenosine in the loop that is critical for high-affinity ligand binding. NEO2A was obtained from the same selection and, although they are only 43% identical in overall sequence, NEO1A and NEO2A share similar loop sequences. Experimental analysis by 1D NMR and 2-aminopurine reporters combined with molecular dynamics modeling revealed similar structural and dynamical characteristics in both aptamers. These results are consistent with the hypothesis that the target ligand...
Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution
Proceedings of the National Academy of Sciences, 2013
Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5′ and 3′ ends of the aptamer domain, adopts a predominantly folded structure in the presence o...
Mutational Analysis of the Purine Riboswitch Aptamer Domain †
Biochemistry, 2007
The purine riboswitch is one of a number of mRNA elements commonly found in the 5′-untranslated region capable of controlling expression in a cis-fashion via its ability to directly bind small molecule metabolites. Extensive biochemical and structural analysis of the nucleobase-binding domain of the riboswitch, referred to as the aptamer domain, has revealed that the mRNA recognizes its cognate ligand using an intricately folded three-way junction motif that completely encapsulates the ligand. High affinity binding of the purine nucleobase is facilitated by a distal loop-loop interaction that is conserved between both the adenine and guanine riboswitches. To understand the contribution of conserved nucleotides in both the three-way junction and the loop-loop interaction of this RNA we performed a detailed mutagenic survey of these elements in the context of an adenine-responsive variant of the xpt-pbuX guanine riboswitch from B. subtilis. The varying ability of these mutants to bind ligand as measured by isothermal titration calorimetry (ITC), uncovered the conserved nucleotides whose identity is required for purine binding. Crystallographic analysis of the bound form of five mutants and chemical probing of their free state demonstrate that the identity of several universally conserved nucleotides are not essential for formation of the RNA-ligand complex but rather for maintaining a binding-competent form of the free RNA. These data show that conservation patterns in riboswitches arise from a combination of formation of the ligand-bound complex, promoting an open form of the free RNA, and participating in the secondary structural switch with the expression platform.
Journal of Molecular Graphics & Modelling, 2011
Riboswitches are mRNA structural elements that act as intracellular sensors of small-molecule metabolites. By undergoing conformational changes capable of modulating translation or terminating transcription, riboswitches are able to play a role in regulating the concentration of essential metabolites in the cell. Computer-guided fluorescence experiments were carried out to interrogate molecular dynamics and conformational changes in the minimal riboswitch aptamer that