The importance of helix P1 stability for structural pre-organization and ligand binding affinity of the adenine riboswitch aptamer domain (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.
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.
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
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.
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
Nucleic Acids Research, 2007
Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg 2+ -ions. Furthermore, a role for Mg 2+ -ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNAadenine complex in solution as studied by highresolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg 2+ , but becomes partially pre-organized for ligand binding in the presence of Mg 2+ . Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg 2+ is more pronounced.
Cooperative and Directional Folding of the preQ 1 Riboswitch Aptamer Domain
Journal of the American Chemical Society, 2011
Riboswitches are cis-acting RNA fragments that regulate gene expression by sensing cellular levels of the associated small metabolites. In bacteria, the class I preQ 1 riboswitch allows the fine-tuning of queuosine biosynthesis in response to the intracellular concentration of the queuosine anabolic intermediate preQ 1 . When binding preQ 1 , the aptamer domain undergoes a significant degree of secondary and tertiary structural rearrangement and folds into an H-type pseudoknot. Conformational "switching" of the riboswitch aptamer domain upon recognizing its cognate metabolite plays a key role in the regulatory mechanism of the preQ 1 riboswitch. We investigate the folding mechanism of the preQ 1 riboswitch aptamer domain using all-atom Go hmodel simulations. The folding pathway of such a single domain is found to be cooperative and sequentially coordinated, as the folding proceeds in the 5 0 f 3 0 direction. This kinetically efficient folding mechanism suggests a fast ligandbinding response in competition with RNA elongation.
Proceedings of the National Academy of Sciences, 2012
Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues. gene regulation | translational control | metabolism R NA structures fulfill important roles in the regulation of gene expression including the control of translational initiation, transcriptional termination, and alternative splicing (1-5). In many cases, an RNA conformational change is crucial to gene regulation since one RNA sequence may adopt alternate structures. Regulation of gene expression is achieved when an input domain senses varying cellular conditions, resulting in shifting the balance between two or more structural states.
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...
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.