FRET-guided modeling of nucleic acids.

The functional diversity of RNAs is encoded in their innate conformational heterogeneity. The combination of single-molecule spectroscopy and computational modeling offers new attractive opportunities to map structural transitions within nucleic acid ensembles. Here, we describe a framework to harmonize single-molecule Förster resonance energy transfer (FRET) measurements with molecular dynamics simulations and de novo structure prediction. Using either all-atom or implicit fluorophore modeling, we recreate FRET experiments in silico, visualize the underlying structural dynamics and quantify the reaction coordinates. Using multiple accessible-contact volumes as a post hoc scoring method for fragment assembly in Rosetta, we demonstrate that FRET can be used to filter a de novo RNA structure prediction ensemble by refuting models that are not compatible with in vitro FRET measurement. We benchmark our FRET-assisted modeling approach on double-labeled DNA strands and validate it against an intrinsically dynamic manganese(II)-binding riboswitch. We show that a FRET coordinate describing the assembly of a four-way junction allows our pipeline to recapitulate the global fold of the riboswitch displayed by the crystal structure. We conclude that computational fluorescence spectroscopy facilitates the interpretability of dynamic structural ensembles and improves the mechanistic understanding of nucleic acid interactions.

Corrin Ring Modifications Reveal the Chemical and Spatial Requirements for the B12-btuB Riboswitch Interaction.

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i.e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.

From Enigma to Revelation: Unravelling Biological Functions of Ubiquitous Small Ribozymes.

RNA, widely recognized as an information-carrier molecule, is capable of catalyzing essential biological processes through ribozymes. Despite their ubiquity, specific functions in a biological context and phenotypes based on the ribozymes' activity are often unknown. Here, we present the discovery of a subgroup of minimal HDV-like ribozymes, which reside 3' to viral tRNAs and appear to cleave the 3'-trailers of viral premature tRNA transcripts. This proposed tRNA-processing function is unprecedented for any ribozymes, thus, we designate this subgroup as theta ribozymes. Most theta ribozymes were identified in Caudoviricetes bacteriophages, the main constituent (>90%) of the mammalian gut virome. Intriguingly, our findings further suggest the involvement of theta ribozymes in the transition of certain bacteriophages between distinct genetic codes, thus possibly contributing to the phage lysis trigger. Our discovery expands the limited repertoire of biological functions attributed to HDV-like ribozymes and provides insights into the fascinating world of RNA catalysis.

Organometallic Pillarplexes That Bind DNA 4-Way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination, and repair) and are dynamic structures that adopt either open or closed conformations, the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core, an ideal structure to interact with open DNA junction cavities. Combining experimental studies and MD simulations, we show that an Au pillarplex can bind DNA 4-way (Holliday) junctions in their open form, a binding mode not accessed by synthetic agents before. Pillarplexes can bind 3-way junctions too, but their large size leads them to open up and expand that junction, disrupting the base pairing, which manifests in an increased hydrodynamic size and lower junction thermal stability. At high loading, they rearrange both 4-way and 3-way junctions into Y-shaped forks to increase the available junction-like binding sites. Isostructural Ag pillarplexes show similar DNA junction binding behavior but lower solution stability. This pillarplex binding contrasts with (but complements) that of metallo-supramolecular cylinders, which prefer 3-way junctions and can rearrange 4-way junctions into 3-way junction structures. The pillarplexes' ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures. In human cells, the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher-order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction binders into organometallic chemistry.

Group II Introns: Highly Structured yet Dynamic.

RNA splicing, the removal of introns and ligation of exons, is a crucial process during mRNA maturation. Group II introns are large ribozymes that self-catalyze their splicing, as well as their transposition. They are living fossils of spliceosomal introns and eukaryotic retroelements. The yeast mitochondrial Sc.ai5γ is the first identified and best-studied self-splicing group II intron. A combination of biochemical, biophysical, and computational tools enables studying its catalytic properties, structure, and dynamics, while also serving to develop new therapeutic and biotechnological tools. We survey the history of group II intron studies paralleling the trends in RNA methodology with Sc.ai5γ in the spotlight.

FRETraj: integrating single-molecule spectroscopy with molecular dynamics.

SUMMARY: Quantitative interpretation of single-molecule FRET experiments requires a model of the dye dynamics to link experimental energy transfer efficiencies to distances between atom positions. We have developed FRETraj, a Python module to predict FRET distributions based on accessible-contact volumes (ACV) and simulated photon statistics. FRETraj helps to identify optimal fluorophore positions on a biomolecule of interest by rapidly evaluating donor-acceptor distances. FRETraj is scalable and fully integrated into PyMOL and the Jupyter ecosystem. Here, we describe the conformational dynamics of a DNA hairpin by computing multiple ACVs along a molecular dynamics trajectory and compare the predicted FRET distribution with single-molecule experiments. FRET-assisted modeling will accelerate the analysis of structural ensembles in particular dynamic, non-coding RNAs and transient protein-nucleic acid complexes. AVAILABILITY AND IMPLEMENTATION: FRETraj is implemented as a cross-platform Python package available under the GPL-3.0 on Github (https://github.com/RNA-FRETools/fretraj) and is documented at https://RNA-FRETools.github.io/fretraj. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Coordination Chemistry of Nucleotides and Antivirally Active Acyclic Nucleoside Phosphonates, including Mechanistic Considerations.

Considering that practically all reactions that involve nucleotides also involve metal ions, it is evident that the coordination chemistry of nucleotides and their derivatives is an essential corner stone of biological inorganic chemistry. Nucleotides are either directly or indirectly involved in all processes occurring in Nature. It is therefore no surprise that the constituents of nucleotides have been chemically altered-that is, at the nucleobase residue, the sugar moiety, and also at the phosphate group, often with the aim of discovering medically useful compounds. Among such derivatives are acyclic nucleoside phosphonates (ANPs), where the sugar moiety has been replaced by an aliphatic chain (often also containing an ether oxygen atom) and the phosphate group has been replaced by a phosphonate carrying a carbon-phosphorus bond to make the compounds less hydrolysis-sensitive. Several of these ANPs show antiviral activity, and some of them are nowadays used as drugs. The antiviral activity results from the incorporation of the ANPs into the growing nucleic acid chain-i.e., polymerases accept the ANPs as substrates, leading to chain termination because of the missing 3'-hydroxyl group. We have tried in this review to describe the coordination chemistry (mainly) of the adenine nucleotides AMP and ATP and whenever possible to compare it with that of the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA2- = adenine(N9)-CH2-CH2-O-CH2-PO32) [or its diphosphate (PMEApp4-)] as a representative of the ANPs. Why is PMEApp4- a better substrate for polymerases than ATP4-? There are three reasons: (i) PMEA2- with its anti-like conformation (like AMP2-) fits well into the active site of the enzyme. (ii) The phosphonate group has an enhanced metal ion affinity because of its increased basicity. (iii) The ether oxygen forms a 5-membered chelate with the neighboring phosphonate and favors thus coordination at the Pα group. Research on ANPs containing a purine residue revealed that the kind and position of the substituent at C2 or C6 has a significant influence on the biological activity. For example, the shift of the (C6)NH2 group in PMEA to the C2 position leads to 9-[2-(phosphonomethoxy)ethyl]-2-aminopurine (PME2AP), an isomer with only a moderate antiviral activity. Removal of (C6)NH2 favors N7 coordination, e.g., of Cu2+, whereas the ether O atom binding of Cu2+ in PMEA facilitates N3 coordination via adjacent 5- and 7-membered chelates, giving rise to a Cu(PMEA)cl/O/N3 isomer. If the metal ions (M2+) are M(α,ß)-M(γ)-coordinated at a triphosphate chain, transphosphorylation occurs (kinases, etc.), whereas metal ion binding in a M(α)-M(ß,γ)-type fashion is relevant for polymerases. It may be noted that with diphosphorylated PMEA, (PMEApp4-), the M(α)-M(ß,γ) binding is favored because of the formation of the 5-membered chelate involving the ether O atom (see above). The self-association tendency of purines leads to the formation of dimeric [M2(ATP)]2(OH)- stacks, which occur in low concentration and where one half of the molecule undergoes the dephosphorylation reaction and the other half stabilizes the structure-i.e., acts as the "enzyme" by bridging the two ATPs. In accord herewith, one may enhance the reaction rate by adding AMP2- to the [Cu2(ATP)]2(OH)- solution, as this leads to the formation of mixed stacked Cu3(ATP)(AMP)(OH)- species, in which AMP2- takes over the structuring role, while the other "half" of the molecule undergoes dephosphorylation. It may be added that Cu3(ATP)(PMEA) or better Cu3(ATP)(PMEA)(OH)- is even a more reactive species than Cu3(ATP)(AMP)(OH)-. - The matrix-assisted self-association and its significance for cell organelles with high ATP concentrations is summarized and discussed, as is, e.g., the effect of tryptophanate (Trp-), which leads to the formation of intramolecular stacks in M(ATP)(Trp)3- complexes (formation degree about 75%). Furthermore, it is well-known that in the active-site cavities of enzymes the dielectric constant, compared with bulk water, is reduced; therefore, we have summarized and discussed the effect of a change in solvent polarity on the stability and structure of binary and ternary complexes: Opposite effects on charged O sites and neutral N sites are observed, and this leads to interesting insights.

Magnesium(II)-ATP Complexes in 1-Ethyl-3-Methylimidazolium Acetate Solutions Characterized by 31 Mg ß-Radiation-Detected NMR Spectroscopy.

The complexation of MgII with adenosine 5'-triphosphate (ATP) is omnipresent in biochemical energy conversion, but is difficult to interrogate directly. Here we use the spin- 1/2 ß-emitter 31 Mg to study MgII -ATP complexation in 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) solutions using ß-radiation-detected nuclear magnetic resonance (ß-NMR). We demonstrate that (nuclear) spin-polarized 31 Mg, following ion-implantation from an accelerator beamline into EMIM-Ac, binds to ATP within its radioactive lifetime before depolarizing. The evolution of the spectra with solute concentration indicates that the implanted 31 Mg initially bind to the solvent acetate anions, whereafter they undergo dynamic exchange and form either a mono- (31 Mg-ATP) or di-nuclear (31 MgMg-ATP) complex. The chemical shift of 31 Mg-ATP is observed up-field of 31 MgMg-ATP, in accord with quantum chemical calculations. These observations constitute a crucial advance towards using ß-NMR to probe chemistry and biochemistry in solution.

Screening for potential interaction partners with surface plasmon resonance imaging coupled to MALDI mass spectrometry.

We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5γ) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5γ (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo.

NMR solution structure of tricyclo-DNA containing duplexes: insight into enhanced thermal stability and nuclease resistance.

Tc-DNA is a conformationally constrained oligonucleotide analogue which shows significant increase in thermal stability when hybridized with RNA, DNA or tc-DNA. Remarkably, recent studies revealed that tc-DNA antisense oligonucleotides (AO) hold great promise for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy. To date, no high-resolution structural data is available for fully modified tc-DNA duplexes and little is known about the origins of their enhanced thermal stability. Here, we report the structures of a fully modified tc-DNA oligonucleotide paired with either complementary RNA, DNA or tc-DNA. All three investigated duplexes maintain a right-handed helical structure with Watson-Crick base pairing and overall geometry intermediate between A- and B-type, but closer to A-type structures. All sugars of the tc-DNA and RNA residues adopt a North conformation whereas the DNA deoxyribose are found in a South-East-North conformation equilibrium. The conformation of the tc-DNA strand in the three determined structures is nearly identical and despite the different nature and local geometry of the complementary strand, the overall structures of the examined duplexes are very similar suggesting that the tc-DNA strand dominates the duplex structure.

Optimal molecular crowding accelerates group II intron folding and maximizes catalysis.

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an "optimum" [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.

Site-specific dual-color labeling of long RNAs for single-molecule spectroscopy.

Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3'-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry. We validate this modular labeling platform using a regulatory RNA of 275 nucleotides, the btuB riboswitch of Escherichia coli, demonstrate its general applicability by modifying a base within a duplex, and show its site-selectivity in targeting a pair of adjacent adenines. Native folding and function of the RNA is confirmed on the single-molecule level by using FRET as a sensor to visualize and characterize the conformational equilibrium of the riboswitch upon binding of its cofactor adenosylcobalamin. The presented labeling strategy overcomes size and site constraints that have hampered routine production of labeled RNA that are beyond 200 nt in length.

The Bioinorganic Periodic Table.

Life depends on metals. While carbon, in terms of abundance and versatility, is considered THE element of life, the vast variety and diversity of the chemistry taking place in living organisms could not be achieved without metal ions. More than twenty metals are found in the human body, most of them being essential, some beneficial, and for others it is still unknown what role they might fulfil in a living cell. Here we give a short introduction into the bioinorganic world of the periodic table, providing just a few examples of key metals for life and aiming to give a flavour to gain further insights into this exciting field of inorganic chemistry at the intersection to the life sciences.

Stick, Flick, Click: DNA-guided Fluorescent Labeling of Long RNA for Single-molecule FRET.

Exploring the spatiotemporal dynamics of biomolecules on a single-molecule level requires innovative ways to make them spectroscopically visible. Fluorescence resonance energy transfer (FRET) uses a pair of organic dyes as reporters to measure distances along a predefined biomolecular reaction coordinate. For this nanoscopic ruler to work, the fluorescent labels need to be coupled onto the molecule of interest in a bioorthogonal and site-selective manner. Tagging large non-coding RNAs with single-nucleotide precision is an open challenge. Here we summarize current strategies in labeling riboswitches and ribozymes for fluorescence spectroscopy and FRET in particular. A special focus lies on our recently developed, DNA-guided approach that inserts two fluorophores through a stepwise process of templated functionality transfer and click chemistry.

Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme.

Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg(2+), the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb(3+)-induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg(2+)ions. Up to eight Mg(2+)ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%-90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg(2+)promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg(2+)binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg(2+)for the nested double pseudoknot structure.

Specific phosphorothioate substitution within domain 6 of a group II intron ribozyme leads to changes in local structure and metal ion binding.

Group II introns are large self-splicing ribozymes that require high amounts of monovalent and divalent metal ions for folding and catalysis under in vitro conditions. Domain 6 of these ribozymes contains a highly conserved adenosine whose 2'-OH acts as a nucleophile during self-cleavage via the branching pathway. We have previously suggested a divalent metal ion that binds to the major groove at the GU wobble pair above the branch-A in a minimal, but active branch domain construct (D6-27) from the yeast mitochondrial intron Sc.ai5γ. Here we characterize metal ion binding to the phosphate oxygens at the branch site. In vitro transcription yielded a D6-27 construct where all R P oxygens of the uridine phosphate groups are replaced by sulfur (α-thio-D6-27). We determined its NMR structure, the second RNA-only structure containing thiophosphate groups. [31P] resonances were assigned and chemical shift changes monitored upon titration with Cd2+. In addition, the two uridines flanking the branch-point, U19 and U21 were specifically thioated by chemical synthesis (thio-U19-D6-27 and thio-U19/U21-D6-27), enabling us to study Cd2+ binding at the R P-, as well as the S P- position of the corresponding phosphate oxygens. Our studies reveal that both non-bridging phosphate oxygens of U19 are involved in metal ion coordination, whereas only the major groove phosphate oxygen of U21 is influenced. Together with NOE data of a hexaamminecobalt(III) titration, this suggests a single metal ion binding site at the GU wobble pair above the branch point in the major groove of D6 of this group II intron ribozyme.

Cation-induced kinetic heterogeneity of the intron-exon recognition in single group II introns.

RNA is commonly believed to undergo a number of sequential folding steps before reaching its functional fold, i.e., the global minimum in the free energy landscape. However, there is accumulating evidence that several functional conformations are often in coexistence, corresponding to multiple (local) minima in the folding landscape. Here we use the 5'-exon-intron recognition duplex of a self-splicing ribozyme as a model system to study the influence of Mg(2+) and Ca(2+) on RNA tertiary structure formation. Bulk and single-molecule spectroscopy reveal that near-physiological M(2+) concentrations strongly promote interstrand association. Moreover, the presence of M(2+) leads to pronounced kinetic heterogeneity, suggesting the coexistence of multiple docked and undocked RNA conformations. Heterogeneity is found to decrease at saturating M(2+) concentrations. Using NMR, we locate specific Mg(2+) binding pockets and quantify their affinity toward Mg(2+). Mg(2+) pulse experiments show that M(2+) exchange occurs on the timescale of seconds. This unprecedented combination of NMR and single-molecule Förster resonance energy transfer demonstrates for the first time to our knowledge that a rugged free energy landscape coincides with incomplete occupation of specific M(2+) binding sites at near-physiological M(2+) concentrations. Unconventional kinetics in nucleic acid folding frequently encountered in single-molecule experiments are therefore likely to originate from a spectrum of conformations that differ in the occupation of M(2+) binding sites.

Structure determination of noncanonical RNA motifs guided by ¹H NMR chemical shifts.

Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating ¹H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6-2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases.

Mg(2+)-induced conformational changes in the btuB riboswitch from E. coli.

Mg(2+) 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 Mg(2+) 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 Mg(2+). 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 Mg(2+) (or low Mg(2+) concentration) and the other appearing above ∼0.5 mM Mg(2+). Distinct regions of the riboswitch exhibit different dissociation constants toward Mg(2+), indicating a stepwise folding of the btuB RNA. Increasing the Mg(2+) concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg(2+) 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 Mg(2+) 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 Mg(2+) indicates a crucial role played by Mg(2+) for defining an active conformation of the riboswitch.

NMR structure of the 5' splice site in the group IIB intron Sc.ai5γ--conformational requirements for exon-intron recognition.

A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5' exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5' exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5' exon and the intron. Here, we present the NMR solution structures of the d3' hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg(2+) ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.