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.     

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.     

Modeling the noncovalent interactions at the metabolite binding site in purine riboswitches

We present gas phase quantum chemical studies on the metabolite binding interactions in two important purine riboswitches, the adenine and guanine riboswitches, at the B3LYP/6-31G(d,p) level of theory. In order to gain insights into the strucutral basis of their discriminative abilities of regulating gene expression, the structural properties and binding energies for the gas phase optimized geometries of the metabolite bound binding pocket are analyzed and compared with their respective crystal geometries. Kitaura-Morokuma analysis has been carried out to calculate and decompose the interaction energy into various components. NBO and AIM analysis has been carried out to understand the strength and nature of binding of the individual aptamer bases with their respective purine metabolites. The Y74 base, U in case of adenine riboswitch and C in case of guanine riboswitch constitutes the only differentiating element between the two binding pockets. As expected, with W:W cis G:C74 interaction contributing more than 50% of the total binding energy, the interaction energy for metabolite binding as calculated for guanine (-46.43 Kcal/mol) is nearly double compared to the corresponding value for that of adenine (-24.73 Kcal/mol) in the crystal context. Variations in the optimized geometries for different models and comparison of relative contribution to metabolite binding involving four conserved bases reveal the possible role of U47:U51 W:H trans pair in the conformational transition of the riboswitch from the metabolite free to metabolite bound state. Our results are also indicative of significant contributions from stacking and magnesium ion interactions toward cooperativity effects in metabolite recognition.     

Purine sensing by riboswitches.

Structured mRNA elements called riboswitches control gene expression by binding to small metabolites. Over a dozen riboswitch classes have been characterized that target a broad range of molecules and vary widely in size and secondary structure. Four of the known riboswitch classes recognize purines or modified purines. Three of these classes are closely related in conserved sequence and secondary structure, but members of these classes selectively recognize guanine, adenine or 2'-deoxyguanosine. Members of the fourth riboswitch class adopt a distinct structure to form a selective binding pocket for the guanine analogue preQ(1) (7-aminomethyl-7-deazaguanine). All four classes of purine-sensing riboswitches are most likely to recognize their respective metabolites by utilizing a riboswitch residue to make a canonical Watson-Crick base-pair with the ligand. This review will provide a summary of the purine-sensing riboswitches, as well as discuss the complex functions and applications of these RNAs.     

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.     

Purine sensing by riboswitches

Structured mRNA elements called riboswitches control gene expression by binding to small metabolites. Over a dozen riboswitch classes have been characterized that target a broad range of molecules and vary widely in size and secondary structure. Four of the known riboswitch classes recognize purines or modified purines. Three of these classes are closely related in conserved sequence and secondary structure, but members of these classes selectively recognize guanine, adenine or 2'-deoxyguanosine. Members of the fourth riboswitch class adopt a distinct structure to form a selective binding pocket for the guanine analogue preQ(1) (7-aminomethyl-7-deazaguanine). All four classes of purine-sensing riboswitches are most likely to recognize their respective metabolites by utilizing a riboswitch residue to make a canonical Watson-Crick base-pair with the ligand. This review will provide a summary of the purine-sensing riboswitches, as well as discuss the complex functions and applications of these RNAs.     

Riboswitch structure: an internal residue mimicking the purine ligand

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson–Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39–C65 and A39–U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.     

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.     

Structure and mechanism of purine-binding riboswitches

A riboswitch is a non-protein coding sequence capable of directly binding a small molecule effector without the assistance of accessory proteins to regulate expression of the mRNA in which it is embedded. Currently, over 20 different classes of riboswitches have been validated in bacteria with the promise of many more to come, making them an important means of regulating the genome in the bacterial kingdom. Strikingly, half of the known riboswitches recognize effector compounds that contain a purine or related moiety. In the last decade, significant progress has been made to determine how riboswitches specifically recognize these compounds against the background of many other similar cellular metabolites and transduce this signal into a regulatory response. Of the known riboswitches, the purine family containing guanine, adenine and 2'-deoxyguanosine-binding classes are the most extensively studied, serving as a simple and useful paradigm for understanding how these regulatory RNAs function. This review provides a comprehensive summary of the current state of knowledge regarding the structure and mechanism of these riboswitches, as well as insights into how they might be exploited as therapeutic targets and novel biosensors.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism

Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms.     

Riboswitches that sense S-adenosylmethionine and S-adenosylhomocysteine.

Numerous riboswitches have been discovered that specifically recognize metabolites and modulate gene expression. Each riboswitch class is defined either by the consensus sequence and structural features of its metabolite-binding aptamer domain, or by the distinct metabolite that the aptamer recognizes. Several distinct classes of riboswitches that respond to S-adenosylmethionine (SAM or AdoMet) have been discovered. Representatives of these classes have been shown to strongly discriminate against S-adenosylhomocystenine (SAH or AdoHcy), which is the metabolic byproduct produced when SAM is used as a cofactor for methylation reactions. However, a distinct class of riboswitches that selectively binds SAH, and strongly discriminates against SAM, also has been discovered. Herein we compare the features of SAM and SAH riboswitches, which help showcase the enormous structural diversity that RNA can harness to form precision genetic switches for compounds that are critical for fundamental metabolic processes.     

Further characterization of a depurinated DNA-purine base insertion activity from cultured human fibroblasts.

The purification from cultured human fibroblasts of a protein that binds specifically to partially depurinated DNA and inserts purines into those sites is described. The purine insertion, but not the binding, requires K+. The DNA binding can be saturated with increasing apurinic sites and is weakened by the presence of adenine or guanine. Base insertion into depurinated DNA is specific for adenine or guanine; none is observed with dATP or dGTP. When the depurinated DNA substrate is specifically cleaved with apurinic endonuclease, no purine insertion occurs. Guanine insertion does not occur into tRNA or depyrimidinated DNA, and thymine is not inserted into either depyrimidinated DNA or depurinated DNA. Purine insertion activity follows Michaelis-Menten kinetics with respect to purintes; the apparent Km values for both adenine and guanine are 5 microM. The enzyme binds the purine bases very tightly. Adenine binding saturates at less than 1 microM adenine, perhaps reflecting the low intracellular adenine concentration. The binding protein specific for UV-irradiated DNA (Feldberg, R.S., and Grossman, L. (1976) Biochemistry 15, 2402-2408) had no detectable purine or pyrimidine base insertion activity with depurinated or depyrimidinated DNAs.