Structure of a rare non-standard sequence k-turn bound by L7Ae protein

Kt-23 from Thelohania solenopsae is a rare RNA kink turn (k-turn) where an adenine replaces the normal guanine at the 2n position. L7Ae is a member of a strongly conserved family of proteins that bind a range of k-turn structures in the ribosome, box C/D and H/ACA small nucleolar RNAs and U4 small nuclear RNA. We have solved the crystal structure of T. solenopsae Kt-23 RNA bound to Archeoglobus fulgidus L7Ae protein at a resolution of 2.95 Å. The protein binds in the major groove displayed on the outer face of the k-turn, in a manner similar to complexes with standard k-turn structures. The k-turn adopts a standard N3 class conformation, with a single hydrogen bond from A2b N6 to A2n N3. This contrasts with the structure of the same sequence located in the SAM-I riboswitch, where it adopts an N1 structure, showing the inherent plasticity of k-turn structure. This potentially can affect any tertiary interactions in which the RNA participates.     

RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn

The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G?A and A?G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make?a?long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure.     

Structure and folding of a rare, natural kink turn in RNA with an A*A pair at the 2b*2n position

The kink turn (k-turn) is a frequently occurring motif, comprising a bulge followed by G•A and A•G pairs that introduces a sharp axial bend in duplex RNA. Natural k-turn sequences exhibit significant departures from the consensus, including the A•G pairs that form critical interactions stabilizing the core of the structure. Kt-23 found in the small ribosomal subunit differs from the consensus in many organisms, particularly in the second A•G pair distal to the bulge (2b•2n). Analysis of many Kt-23 sequences shows that the frequency of occurrence at the 2n position (i.e., on the nonbulged strand, normally G in standard k-turns) is U>C>G>A. Less than 1% of sequences have A at the 2n position, but one such example occurs in Thelohania solenopsae Kt-23. This sequence folds only weakly in the presence of Mg²⁺ ions but is induced to fold normally by the binding of L7Ae protein. Introduction of this sequence into the SAM-I riboswitch resulted in normal binding of SAM ligand, indicating that tertiary RNA contacts have resulted in k-turn folding. X-ray crystallography shows that the T. solenopsae Kt-23 adopts a standard k-turn geometry, making the key, conserved hydrogen bonds in the core and orienting the 1n (of the bulge-proximal A•G pair) and 2b adenine nucleobases in position facing the opposing minor groove. The 2b and 2n adenine nucleobases are not directly hydrogen bonded, but each makes hydrogen bonds to their opposing strands.     

Folding of the cocaine aptamer studied by EPR and fluorescence spectroscopies using the bifunctional spectroscopic probe Ç

The cocaine aptamer is a DNA molecule that binds cocaine at the junction of three helices. The bifunctional spectroscopic probe Ç was incorporated independently into three different positions of the aptamer and changes in structure and dynamics upon addition of the cocaine ligand were studied. Nucleoside Ç contains a rigid nitroxide spin label and can be studied directly by electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy after reduction of the nitroxide to yield the fluoroside Ç^f. Both the EPR and the fluorescence data for aptamer 2 indicate that helix III is formed before cocaine binding. Upon addition of cocaine, increased fluorescence of a fully base-paired Ç^f, placed at the three-way junction in helix III, was observed and is consistent with a helical tilt from a coaxial stack of helices II and III. EPR and fluorescence data clearly show that helix I is formed upon addition of cocaine, concomitant with the formation of the Y-shaped three-way helical junction. The EPR data indicate that nucleotides in helix I are more mobile than nucleotides in regular duplex regions and may reflect increased dynamics due to the short length of helix I.     

The plasticity of a structural motif in RNA: Structural polymorphism of a kink turn as a function of its environment

The k-turn is a widespread structural motif that introduces a tight kink into the helical axis of double-stranded RNA. The adenine bases of consecutive G•A pairs are directed toward the minor groove of the opposing helix, hydrogen bonding in a typical A-minor interaction. We show here that the available structures of k-turns divide into two classes, depending on whether N3 or N1 of the adenine at the 2b position accepts a hydrogen bond from the O2′ at the −1n position. There is a coordinated structural change involving a number of hydrogen bonds between the two classes. We show here that Kt-7 can adopt either the N3 or N1 structures depending on environment. While it has the N1 structure in the ribosome, on engineering it into the SAM-I riboswitch, it changes to the N3 structure, resulting in a significant alteration in the trajectory of the helical arms.     

Ion-induced folding of a kink turn that departs from the conventional sequence

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G•A and A•G base pairs, and when these G•A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A•G pair is replaced by a non-Watson–Crick A•U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg²⁺ or Na⁺ ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A•G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson–Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A•U pair is more important than the other.     

Measurement of the Change in Twist at a Helical Junction in RNA Using the Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5′ terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter κ² as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.     

Measurement of the Change in Twist at a Helical Junction in RNA Using the?Orientation Dependence of FRET

Indocarbocyanine fluorophores attached via the 5′ terminus of double-stranded nucleic acids have a strong propensity to stack onto the terminal basepair. We previously demonstrated that the efficiency of fluorescence resonance energy transfer between cyanine 3 and 5 terminally attached to duplex species exhibits a pronounced modulation with helix length. This results from a systematic variation in the orientation parameter κ² as the relative rotation of the fluorophore transition moments changes due to the helical geometry. Analysis of such profiles provides a rich source of orientational information. In this work, we applied this methodology to the structure of a three-way helical junction that plays an important role in the hepatitis C virus internal ribosome entry site. By comparing matched pairs of duplex and junction species, we were able to measure the change in rotation at the junction. The data reveal a 29.5° overwinding and a small axial extension. This shows the power of this approach for measuring orientational information in biologically important RNA junctions.     

Definitions and analysis of DNA Holliday junction geometry

A number of single-crystal structures have now been solved of the four-stranded antiparallel stacked-X form of the Holliday junction. These structures demonstrate how base sequence, substituents, and drug and ion interactions affect the general conformation of this recombination intermediate. The geometry of junctions had previously been described in terms of a specific set of parameters that include: (i) the angle relating the ends of DNA duplexes arms of the junction (interduplex angle); (ii) the relative rotation of the duplexes about the helix axes of the stacked duplex arms (J_roll); and (iii) the translation of the duplexes along these helix axes (J_slide). Here, we present a consistent set of definitions and methods to accurately calculate each of these parameters based on the helical features of the stacked duplex arms in the single-crystal structures of the stacked-X junction, and demonstrate how each of these parameters contributes to an overall conformational feature of the structure. We show that the values for these parameters derived from global rather than local helical axes through the stacked bases of the duplex arms are the most representative of the stacked-X junction conformation. In addition, a very specific parameter (J_twist) is introduced which relates the relative orientation of the stacked duplex arms across the junction which, unlike the interduplex angle, is length independent. The results from this study provide a general means to relate the geometric features seen in the crystal structures to those determined in solution.     

Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch

Glycine riboswitches regulate gene expression by feedback modulation in response to cooperative binding to glycine. Here, we report on crystal structures of the second glycine-sensing domain from the Vibrio cholerae riboswitch in the ligand-bound and unbound states. This domain adopts a three-helical fold that centers on a three-way junction and accommodates glycine within a bulge-containing binding pocket above the junction. Glycine recognition is facilitated by a pair of bound Mg(2+) cations and governed by specific interactions and shape complementarity with the pocket. A conserved adenine extrudes from the binding pocket and intercalates into the junction implying that glycine binding in the context of the complete riboswitch could impact on gene expression by stabilizing the riboswitch junction and regulatory P1 helix. Analysis of riboswitch interactions in the crystal and footprinting experiments indicates that adjacent glycine-sensing modules of the riboswitch could form specific interdomain interactions, thereby potentially contributing to the cooperative response.     

Structure of the three-way helical junction of the hepatitis C virus IRES element

The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS₄ junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS₁HS₃ and 2HS₂HS₂ junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg²⁺ ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.     

Conformational changes in the expression domain of the Escherichia coli thiM riboswitch

The thiM riboswitch contains an aptamer domain that adaptively binds the coenzyme thiamine pyrophosphate (TPP). The binding of TPP to the aptamer domain induces structural rearrangements that are relayed to a second domain, the so-called expression domain, thereby interfering with gene expression. The recently solved crystal structures of the aptamer domains of the thiM riboswitches in complex with TPP revealed how TPP stabilizes secondary and tertiary structures in the RNA ligand complex. To understand the global modes of reorganization between the two domains upon metabolite binding the structure of the entire riboswitch in presence and absence of TPP needs to be determined. Here we report the secondary structure of the entire thiM riboswitch from Escherichia coli in its TPP-free form and its transition into the TPP-bound variant, thereby depicting domains of the riboswitch that serve as communication links between the aptamer and the expression domain. Furthermore, structural probing provides an explanation for the lack of genetic control exerted by a riboswitch variant with mutations in the expression domain that still binds TPP.     

Functional equivalence of the uridine turn and the hairpin as building blocks of tertiary structure in the Neurospora VS ribozyme.

Mutational, kinetic, and chemical modification experiments show that one of the three-way helical junctions in the Neurospora VS ribozyme contains a uridine turn that is important for organizing the functional three-dimensional structure of this junction. Disruption of the uridine turn disrupts the structure of the junction and decreases the self-cleavage activity of the ribozyme; however, substitution of the uridine turn with a variety of different hairpins, thereby transforming the three-way junction into a four-way junction, maintains catalytic activity. Chemical modification structure probing reveals that both the native junction and the hairpin-containing junction support the same tertiary interactions required elsewhere in the ribozyme for catalysis. These observations show that functionally equivalent three-dimensional RNA structures can be built from different secondary structure elements.     

SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch

S-box (SAM-I) riboswitches are a widespread class of riboswitches involved in the regulation of sulfur metabolism in Gram-positive bacteria. We report here the 3.0-Å crystal structure of the aptamer domain of the Bacillus subtilis yitJ S-box (SAM-I) riboswitch bound to S-adenosyl-l-methionine (SAM). The RNA folds into two sets of helical stacks spatially arranged by tertiary interactions including a K-turn and a pseudoknot at a four-way junction. The tertiary structure is further stabilized by metal coordination, extensive ribose zipper interactions, and SAM-mediated tertiary interactions. Despite structural differences in the peripheral regions, the SAM-binding core of the B. subtilis yitJ riboswitch is virtually superimposable with the previously determined Thermoanaerobacter tengcongensis yitJ riboswitch structure, suggesting that a highly conserved ligand-recognition mechanism is utilized by all S-box riboswitches. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing analysis further revealed that the alternative base-pairing element in the expression platform controls the conformational switching process. In the absence of SAM, the apo yitJ aptamer domain folds predominantly into a pre-binding conformation that resembles, but is not identical with, the SAM-bound state. We propose that SAM enters the ligand-binding site through the “J1/2-J3/4” gate and “locks” down the SAM-bound conformation through an induced-fit mechanism. Temperature-dependent SHAPE revealed that the tertiary interaction-stabilized SAM-binding core is extremely stable, likely due to the cooperative RNA folding behavior. Mutational studies revealed that certain modifications in the SAM-binding region result in loss of SAM binding and constitutive termination, which suggests that these mutations lock the RNA into a form that resembles the SAM-bound form in the absence of SAM.     

Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch

The thi-box riboswitch regulates gene expression in response to the intracellular concentration of thiamine pyrophosphate (TPP) in archaea, bacteria, and eukarya. To complement previous biochemical, genetic, and structural studies of this phylogenetically widespread RNA domain, we have characterized its interaction with TPP by isothermal titration calorimetry. This shows that TPP binding is highly dependent on Mg²⁺ concentration. The dissociation constant decreases from ∼200 nM at 0.5 mM Mg²⁺ concentration to ∼9 nM at 2.5 mM Mg²⁺ concentration. Binding is enthalpically driven, but the unfavorable entropy of binding decreases as Mg²⁺ concentration rises, suggesting that divalent cations serve to pre-organize the RNA. Mutagenesis, biochemical analysis, and a new crystal structure of the riboswitch suggest that a critical element that participates in organizing the riboswitch structure is the tertiary interaction formed between the P3 and L5 regions. This tertiary contact is distant from the TPP binding site, but calorimetric analysis reveals that even subtle mutations in L5 can have readily detectable effects on TPP binding. The thermodynamic signatures of these mutations, namely decreased favorable enthalpy of binding and small effects on entropy of binding, are consistent with the P3–L5 association contributing allosterically to TPP-induced compaction of the RNA.