Guanine Riboswitch Research Articles

A Structural Basis for the Recognition of 2′-Deoxyguanosine by the Purine Riboswitch

Riboswitches are noncoding RNA elements that are commonly found in the 5′-untranslated region of bacterial mRNA. Binding of a small-molecule metabolite to the riboswitch aptamer domain guides the folding of the downstream sequence into one of two mutually exclusive secondary structures that directs gene expression. The purine riboswitch family, which regulates aspects of purine biosynthesis and transport, contains three distinct classes that specifically recognize guanine/hypoxanthine, adenine, or 2′-deoxyguanosine (dG). Structural analysis of the guanine and adenine classes revealed a binding pocket that almost completely buries the nucleobase within the core of the folded RNA. Thus, it is somewhat surprising that this family of RNA elements also recognizes dG. We have used a combination of structural and biochemical techniques to understand how the guanine riboswitch could be converted into a dG binder and the structural basis for dG recognition. These studies reveal that a limited number of sequence changes to a guanine-sensing RNA are required to cause a specificity switch from guanine to 2′-deoxyguanosine, and to impart an altered structure for accommodating the additional deoxyribose sugar moiety.

RNase P cleaves the adenine riboswitch and stabilizes pbuE mRNA in Bacillus subtilis

RNase P from Bacillus subtilis cleaves in vitro the adenine riboswitch upstream of pbuE, which codes for an adenine efflux pump. The guanine riboswitch, encoded upstream of xpt-pbuX operon, is not cleaved. The cleavage sites do not occur at any predicted structures that should be recognized by RNase P in the theoretical model of the adenine riboswitch. However, it is possible to draw alternative secondary structure models that match the apparent requirements for RNase P substrates at these cleavage sites. Support for these models is provided by appropriate mutagenesis experiments. Adenine showed no effect on the cleavage in vitro of the pbuE adenine riboswitch by RNase P holoenzyme from B. subtilis. The results of genetic experiments performed in B. subtilis support the cleavage of adenine riboswitch by RNase P in vivo and suggest that it induces the stabilization of pbuE mRNA under normal conditions.

Mutational Analysis of the Purine Riboswitch Aptamer Domain

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 Bacillus subtilis. The varying ability of these mutants to bind ligand as measured by isothermal titration calorimetry 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 is 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.

Guanine riboswitch variants from Mesoplasma florum selectively recognize 2′-deoxyguanosine

Several mRNA aptamers have been identified in Mesoplasma florum that have sequence and structural features resembling those of guanine and adenine riboswitches. Two features distinguish these RNAs from established purine-sensing riboswitches. All possess shortened hairpin-loop sequences expected to alter tertiary contacts known to be critical for aptamer folding. The RNAs also carry nucleotide changes in the core of each aptamer that otherwise is strictly conserved in guanine and adenine riboswitches. Some aptamers retain the ability to selectively bind guanine or adenine despite these mutations. However, one variant type exhibits selective and high-affinity binding of 2'-deoxyguanosine, which is consistent with its occurrence in the 5' untranslated region of an operon containing ribonucleotide reductase genes. The identification of riboswitch variants that bind nucleosides and reject nucleobases reveals that natural metabolite-sensing RNA motifs can accrue mutations that expand the diversity of ligand detection in bacteria.

Ligand recognition determinants of guanine riboswitches

Guanine riboswitches negatively modulate transcription upon guanine binding. The aptamer domain is organized around a three-way junction which forms the ligand binding site. Using currently available 89 guanine aptamer sequences, a consensus secondary structure is deduced and reveals differences from the previously identified aptamer consensus. Three positions are found to display different nucleotide requirements. Using a 2-aminopurine binding assay, we show that variations are allowed depending on the aptamer context. However, changes at position 48 markedly decrease ligand binding in a context-independent fashion. This is consistent with previous observations with the adenine riboswitch in which position 48 was proposed to interact with position 74, which normally base pairs with the ligand. The in vivo transcriptional control of endogenous Bacillus subtilis guanine riboswitches was studied using RT-qPCR assays. The ratio of elongated/terminated transcripts is decreased in presence of a high concentration of guanine but is dependent on the riboswitch analyzed. In general, the aptamer-2AP complex affinity correlates well with the in vivo regulation efficiency of the corresponding riboswitch. These studies suggest that core variations of guanine aptamers are used to produce a spectrum of ligand binding affinities which is used in vivo by host riboswitches to perform gene regulation.

Interplay of 'induced fit' and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch

Riboswitches are highly structured elements in the 5′-untranslated regions (5′-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 long-range base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using high-resolution 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. Mg2+-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 Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.

Thermodynamic and Kinetic Characterization of Ligand Binding to the Purine Riboswitch Aptamer Domain

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.

Comparison of Mode Analyses at Different Resolutions Applied to Nucleic Acid Systems

More than two decades of different types of mode analyses has shown that these techniques can be useful in describing large-scale motions in protein systems. A number of mode analyses are available and include quasiharmonics, classical normal mode, block normal mode, and the elastic network model. Each of these methods has been validated for protein systems and this variety allows researchers to choose the technique that gives the best compromise between computational cost and the level of detail in the calculation. These same techniques have not been systematically tested for nucleic acid systems, however. Given the differences in interactions and structural features between nucleic acid and protein systems, the validity of these techniques in the protein regime cannot be directly translated into validity in the nucleic acid realm. In this work, we investigate the usefulness of the above mode analyses as applied to two RNA systems, i.e., the hammerhead ribozyme and a guanine riboswitch. We show that classical normal-mode analysis can match the magnitude and direction of residue fluctuations from the more detailed, anharmonic technique, quasiharmonic analysis of a molecular dynamics trajectory. The block normal-mode approximation is shown to hold in the nucleic acid systems studied. Only the mode analysis at the lowest level of detail, the elastic network model, produced mixed results in our calculations. We present data that suggest that the elastic network model, with the popular parameterization, is not best suited for systems that do not have a close packed structure; this observation also hints at why the elastic network model has been found to be valid for many globular protein systems. The different behaviors of block normal-mode analysis and the elastic network model, which invoke similar degrees of coarse-graining to the dynamics but use different potentials, suggest the importance of applying a heterogeneous potential function in a robust analysis of the dynamics of biomolecules, especially those that are not closely packed. In addition to these comparisons, we briefly discuss insights into the conformational space available to the hammerhead ribozyme.

Riboswitches: natural SELEXion.

Advances in our knowledge of the structure and chemistry of RNA have been harnessed in the process known as SELEX to develop artificial RNA-based molecules that can act as enzymes and ligand binders performing a wide variety of functions. The discovery of riboswitches, natural RNA aptamers involved in genetic regulation, offers a basis of comparison between the artificial selection and the natural selection of structured RNAs for small-molecule recognition. The guanine riboswitch structural determination allows us to draw conclusions regarding the apparent increased complexity of the riboswitch aptamers compared to their in-vitro-selected cousins.

Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine

Riboswitches are genetic regulatory elements found in the 5' untranslated region of messenger RNA that act in the absence of protein cofactors. They are broadly distributed across bacteria and account for the regulation of more than 2% of all genes in Bacillus subtilis, underscoring their importance in the control of cellular metabolism. The 5' untranslated region of many mRNAs of genes involved in purine metabolism and transport contain a guanine-responsive riboswitch that directly binds guanine, hypoxanthine or xanthine to terminate transcription. Here we report the crystal structure at 1.95 A resolution of the purine-binding domain of the guanine riboswitch from the xpt-pbuX operon of B. subtilis bound to hypoxanthine, a prevalent metabolite in the bacterial purine salvage pathway. This structure reveals a complex RNA fold involving several phylogenetically conserved nucleotides that create a binding pocket that almost completely envelops the ligand. Hypoxanthine functions to stabilize this structure and to promote the formation of a downstream transcriptional terminator element, thereby providing a mechanism for directly repressing gene expression in response to an increase in intracellular concentrations of metabolite.

Adenine riboswitches and gene activation by disruption of a transcription terminator.

A class of riboswitches that recognizes guanine and discriminates against other purine analogs was recently identified. RNAs that carry the consensus sequence and structural features of guanine riboswitches are located in the 5' untranslated region (UTR) of numerous prokaryotic genes, where they control the expression of proteins involved in purine salvage and biosynthesis. We report that three representatives of this riboswitch class bind adenine with values for apparent dissociation constant (apparent K(d)) that are several orders of magnitude lower than those for binding guanine. Because preference for adenine is attributable to a single nucleotide substitution, the RNA most likely recognizes its ligand by forming a Watson-Crick base pair. In addition, the adenine riboswitch associated with the ydhL gene of Bacillus subtilis functions as a genetic 'on' switch, wherein adenine binding causes a structural rearrangement that precludes formation of an intrinsic transcription terminator stem.