Ligand-dependent folding of the three-way junction in the purine riboswitch

Riboswitches are highly structured cis-acting elements located in the 5'-untranslated region of messenger RNAs that directly bind small molecule metabolites to regulate gene expression. Structural and biochemical studies have revealed riboswitches experience significant ligand-dependent conformational changes that are coupled to regulation. To monitor the coupling of ligand binding and RNA folding within the aptamer domain of the purine riboswitch, we have chemically probed the RNA with N-methylisatoic anhydride (NMIA) over a broad temperature range. Analysis of the temperature-dependent reactivity of the RNA in the presence and absence of hypoxanthine reveals that a limited set of nucleotides within the binding pocket change their conformation in response to ligand binding. Our data demonstrate that a distal loop-loop interaction serves to restrict the conformational freedom of a significant portion of the three-way junction, thereby promoting ligand binding under physiological conditions.     

Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gsw(loop)) in the absence of Mg(2+). However, if Mg(2+) is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gsw(loop) is tunable through variation of the Mg(2+) concentration. We quantitatively describe the influence of distinct Mg(2+) concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.     

Roles of Mg2+ in TPP-dependent riboswitch

We quantified the effect of Mg(2+) on thiamine pyrophosphate (TPP) binding to TPP-dependent thiA riboswitch RNA. The association constant of TPP binding to the riboswitch at 20 degrees C increased from 1.2 x 10(6) to 50 x 10(6) M(-1) as the Mg(2+) concentration increased from 0 to 1 mM. Furthermore, circular dichroic spectra under various conditions showed that 1 mM Mg(2+) induced a local structural change of the riboswitch, which might be pivotal for TPP binding. These results indicate that a physiological concentration of Mg(2+) can regulate TPP binding to the thiA riboswitch.     

Mg2+-induced tRNA folding

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.     

Navigating the RNA folding landscape

Analysis of individual RNA folding reactions reveals that, as in proteins, cooperative interactions selectively drive RNA toward its biologically active, native conformation. This new work establishes a platform for future investigations of the physical principles underlying the assembly of large RNA enzymes.     

Importance of partially unfolded conformations for Mg(2+)-induced folding of RNA tertiary structure: structural models and free energies of Mg2+ interactions

RNA molecules in monovalent salt solutions generally adopt a set of partially folded conformations containing only secondary structure, the intermediate or I state. Addition of Mg2+ strongly stabilizes the native tertiary structure (N state) relative to the I state. In this paper, a combination of experimental and computational approaches is used to estimate the free energy of the interaction of Mg2+ with partially folded I state RNAs and to consider the possibility that Mg2+ favors "compaction" of the I state to a set of conformations with a higher average charge density. A sequence variant with a drastically destabilized tertiary structure was used as a mimic of I state RNA; as measured by small-angle X-ray scattering, it adopted a progressively more compact conformation over a wide Mg2+ concentration range. Average free energies of the interaction of Mg2+ with the I state mimic were obtained by a fluorescence titration method. To interpret these experimental data further, we generated molecular models of the I state and used them in calculations with the nonlinear Poisson-Boltzmann equation to estimate the change in Mg2+-RNA interaction free energy as the average I state dimensions decrease from expanded to compact. The same models were also used to reproduce quantitatively the experimental difference in excess Mg2+ between N and I states. On the basis of these experiments and calculations, I state compaction appears to enhance Mg2+-I state interaction free energies by 10-20%, but this enhancement is at most 5% of the overall Mg2+-associated stabilization free energy for this rRNA fragment.     

Fast protein folding on downhill energy landscape

Proteins fold in a time range of microseconds to minutes despite the large amount of possible conformers. Molecular dynamics simulations of a three-stranded antiparallel beta-sheet peptide (for a total of 12.6 microsec and 72 folding events) show that at the melting temperature the unfolded state ensemble contains many more conformers than those sampled during a folding event.     

Mg2+-induced conformational changes in the btuB riboswitch from E. coli

Mg²⁺ has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B₁₂ (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B₁₂ transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg²⁺ and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg²⁺. With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg²⁺ (or low Mg²⁺ concentration) and the other appearing above ∼0.5 mM Mg²⁺. Distinct regions of the riboswitch exhibit different dissociation constants toward Mg²⁺, indicating a stepwise folding of the btuB RNA. Increasing the Mg²⁺ concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg²⁺ defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B₁₂, and switch the RNA conformation. Moreover, raising the Mg²⁺ concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg²⁺ indicates a crucial role played by Mg²⁺ for defining an active conformation of the riboswitch.     

Mg2+-dependent folding of a large ribozyme without kinetic traps

The folding kinetics of the catalytic domain of Bacillus subtilis ribonuclease P is analyzed here by fluorescence and catalytic activity. The folding pathway is apparently free of kinetic traps, as indicated by a decrease in folding rates upon the addition of urea. We apply Mg2+ and urea chevron analysis to fully describe the folding and unfolding kinetics of this ribozyme. A folding scheme containing two kinetic intermediates completely accounts for the free energy, the Mg2+ Hill coefficient and the surface buried in the equilibrium transition. At saturating Mg 2+concentrations, folding is limited by a barrier that is independent of Mg2+ and urea. These results describe the first trap-free folding pathway of a large ribozyme and indicate that kinetic traps are not an obligate feature of RNA folding.     

Conformational coupling of Mg2+ and Ca2+ on the three-state folding of calexcitin B

Calexcitin (CE) is a calcium sensor protein that has been implicated in associative learning through the Ca(2+)-dependent inhibition of K(+) channels and activation of ryanodine receptors. CE(B), the major CE variant, was identified as a member of the sarcoplasmic Ca(2+) binding protein family: proteins that can bind both Ca(2+) and Mg(2+). We have now determined the intrinsic Ca(2+) and Mg(2+) binding affinities of CE(B) and investigated their interplay on the folding and structure of CE(B). We find that urea denaturation of CE(B) displays a three-state unfolding transition consistent with the presence of two structural domains. Through a combination of spectroscopic and denaturation studies we find that one domain likely possesses molten globule structure and contains a mixed Ca(2+)/Mg(2+) binding site and a Ca(2+) binding site with weak Mg(2+) antagonism. Furthermore, ion binding to the putative molten globule domain induces native structure formation. The other domain contains a single Ca(2+)-specific binding site and has native structure, even in the absence of ion binding. Ca(2+) binding to CE(B) induces the formation of a recessed hydrophobic pocket. On the basis of measured ion binding affinities and intracellular ion concentrations, it appears that Mg(2+)-CE(B) represents the resting state and Ca(2+)-CE(B) corresponds to the active state, under physiological conditions.     

Finding the path in an RNA folding landscape

In this issue of Structure, Reymond et?al. (2010) combine molecular and computational biology approaches to provide structural details for intermediates in the folding pathway of the hepatitis delta virus ribozyme.     

Morphogenesis of a protein: folding pathways and the energy landscape

Current knowledge on the reaction whereby a protein acquires its native three-dimensional structure was obtained by and large through characterization of the folding mechanism of simple systems. Given the multiplicity of amino acid sequences and unique folds, it is not so easy, however, to draw general rules by comparing folding pathways of different proteins. In fact, quantitative comparison may be jeopardized not only because of the vast repertoire of sequences but also in view of a multiplicity of structures of the native and denatured states. We have tackled the problem of the relationships between the sequence information and the folding pathway of a protein, using a combination of kinetics, protein engineering and computational methods, applied to relatively simple systems. Our strategy has been to investigate the folding mechanism determinants using two complementary approaches, i.e. (i) the study of members of the same family characterized by a common fold, but substantial differences in amino acid sequence, or (ii) heteromorphic pairs characterized by largely identical sequences but with different folds. We discuss some recent data on protein-folding mechanisms by presenting experiments on different members of the PDZ domain family and their circularly permuted variants. Characterization of the energetics and structures of intermediates and TSs (transition states), obtained by Φ-value analysis and restrained MD (molecular dynamics) simulations, provides a glimpse of the malleability of the dynamic states and of the role of the topology of the native states and of the denatured states in dictating folding and misfolding pathways.     

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.     

Protein folding: the free energy surface

Quantitative models and experiments are revealing how the folding free energy surface of a protein is sculpted by sequence and environment. The sometimes conflicting demands of folding, structure and function determine which folding pathways, if any, dominate. Recent advances include experimental estimates of diffusive barrier-crossing times, the observation of ultrafast folders amenable to full-atom simulation, the use of thermodynamic tuning and nonconservative mutations to probe 'hidden' parts of the free energy surface, and a complete microscopic theory of folding.     

Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

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 pre-formation 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 Mg²⁺ 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 Mg²⁺-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 Mg²⁺. 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.     

A rugged free energy landscape separates multiple functional RNA folds throughout denaturation

The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.