Thermodynamic characterization of an engineered tetracycline-binding riboswitch (original) (raw)
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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
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
Riboswitches and synthetic aptamers : a head-to-head comparison
2018
Nucleic acid aptamers are unique molecular structures used for binding to diverse targets. There is a major challenge in adapting in vitro-selected RNA aptamers for building in vivo RNA devices that control cell function. In contrast, their natural nucleic acid counterparts, riboswitches, were deliberately evolved for efficient gene regulation and cellular programming. Encoded within cells, riboswitches exploit a natural aptamer module to bind to an intracellular small molecule target enabling regulation of fundamental metabolic pathways. Here, we review several key features of natural riboswitches that may account for their function in the cellular environment. We compared these features to those of in vitro selected RNA aptamers that bind to small molecule targets. Our analysis revealed that the aptamer structure and magnesium-dependence might be the largest contributors to failed synthetic RNA devices. Thus, we make several suggestions for forthcoming aptamer selections, which ma...
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...
Biochemistry, 2012
The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant, as well as the opening/closing rate constants for the Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (K D) for lysine binding of 1.7(5) mM, and opening/closing rate constants of 1.4(3) s −1 and 0.203(7) s −1 , respectively. These rates predict an apparent dissociation constant for lysine binding (K D, apparent) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports.
Journal of Biological Chemistry, 2011
Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ 1 riboswitch from Thermoanaerobacter tengcongensis in the preQ 1 -bound and free states. Although the mode of preQ 1 recognition is similar to that observed for preQ 0 , surface plasmon resonance revealed an apparent K D of 2.1 ؎ 0.3 nM for preQ 1 but a value of 35.1 ؎ 6.1 nM for preQ 0 . This difference can be accounted for by interactions between the preQ 1 methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ 1 -binding site, resulting in "closed" access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the "closed" free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ 1 riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.
Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer
RNA (New York, N.Y.), 2000
An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 2' deoxyribose substitutions in binding-site residues were tested for theophyllinebinding affinity and the results were compared with the previously determined three-dimensional structure of the RNA-theophylline complex. The RNA-theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2'-deoxy A28 mutant reduces theophylline binding >45-fold at 25 degrees C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 2'-deoxy substitution of U24 also reduces theophylline binding by >90-fold. Several mutations outside the "conserved core" of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absol...
Conditional gene expression by controlling translation with tetracycline-binding aptamers
Nucleic Acids Research, 2003
We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA±metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5¢-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in¯uorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We de®ned nucleotides which in¯uence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only in¯uences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.