Solution structure of a GAAA tetraloop receptor RNA.

The GAAA tetraloop receptor is an 11-nucleotide RNA sequence that participates in the tertiary folding of a variety of large catalytic RNAs by providing a specific binding site for GAAA tetraloops. Here we report the solution structure of the isolated tetraloop receptor as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. The internal loop of the tetraloop receptor has three adenosines stacked in a cross-strand or zipper-like fashion. This arrangement produces a high degree of base stacking within the asymmetric internal loop without extrahelical bases or kinking the helix. Additional interactions within the internal loop include a U. U mismatch pair and a G.U wobble pair. A comparison with the crystal structure of the receptor RNA bound to its tetraloop shows that a conformational change has to occur upon tetraloop binding, which is in good agreement with previous biochemical data. A model for an alternative binding site within the receptor is proposed based on the NMR structure, phylogenetic data and previous crystallographic structures of tetraloop interactions.     

Chrysanthemum chlorotic mottle viroid RNA: dissection of the pathogenicity determinant and comparative fitness of symptomatic and non-symptomatic variants

Chrysanthemum chlorotic mottle viroid (CChMVd) is a small RNA (398-401nt) with hammerhead ribozymes in both polarity strands that mediate self-cleavage of the oligomeric RNA intermediates generated in a rolling-circle mechanism of replication. Within the in vivo branched RNA conformation of CChMVd, a tetraloop has been identified as a major determinant of pathogenicity. Here we present a detailed study of this tetraloop by site-directed mutagenesis, bioassay of the CChMV-cDNA clones and analysis of the resulting progenies. None of the changes introduced in the tetraloop, including its substitution by a triloop or a pentaloop, abolished infectivity. In contrast to observations for other RNAs, the thermodynamically stable GAAA tetraloop characteristic of non-symptomatic CChMVd-NS strains was not functionally interchangeable for other stable tetraloops of the UNCG family, suggesting that the sequence, rather than the structure, is the major factor governing conservation of this motif. In most cases, the changes introduced initially led to symptomless infections, which eventually evolved to be symptomatic concurrently with the prevalence in the progeny of the UUUC tetraloop characteristic of symptomatic CChMVd-S strains. Only in one case did the GAAA tetraloop emerge and eventually dominate the progeny in infected plants that were non-symptomatic. These results revealed two major fitness peaks in the tetraloop (UUUC and GAAA), whose adjacent stem was also under strong selection pressure. Co-inoculations with CChMVd-S and -NS variants showed that only when the latter was in a 100- or 1000-fold excess did the infected plants remain symptomless, confirming the higher biological fitness of the S variant and explaining the lack of symptom expression previously observed in cross-protection experiments.     

Comprehensive features of natural and in vitro selected GNRA tetraloop-binding receptors

Specific recognitions of GNRA tetraloops by small helical receptors are among the most widespread long-range packing interactions in large ribozymes. However, in contrast to GYRA and GAAA tetraloops, very few GNRA/receptor interactions have yet been identified to involve GGAA tetraloops in nature. A novel in vitro selection scheme based on a rigid self-assembling tectoRNA scaffold designed for isolation of intermolecular interactions with A-minor motifs has yielded new GGAA tetraloop-binding receptors with affinity in the nanomolar range. One of the selected receptors is a novel 12 nt RNA motif, (CCUGUG ... AUCUGG), that recognizes GGAA tetraloop hairpin with a remarkable specificity and affinity. Its physical and chemical characteristics are comparable to those of the well-studied '11nt' GAAA tetraloop receptor motif. A second less specific motif (CCCAGCCC ... GAUAGGG) binds GGRA tetraloops and appears to be related to group IC3 tetraloop receptors. Mutational, thermodynamic and comparative structural analysis suggests that natural and in vitro selected GNRA receptors can essentially be grouped in two major classes of GNRA binders. New insights about the evolution, recognition and structural modularity of GNRA and A-minor RNA-RNA interactions are proposed.     

Common and distinctive features of GNRA tetraloops based on a GUAA tetraloop structure at 1.4 A resolution

GNRA tetraloops (N is A, C, G, or U; R is A or G) are basic building blocks of RNA structure that often interact with proteins or other RNA structural elements. Understanding sequence-dependent structural variation among different GNRA tetraloops is an important step toward elucidating the molecular basis of specific GNRA tetraloop recognition by proteins and RNAs. Details of the geometry and hydration of this motif have been based on high-resolution crystallographic structures of the GRRA subset of tetraloops; less is known about the GYRA subset (Y is C or U). We report here the structure of a GUAA tetraloop determined to 1.4 A resolution to better define these details and any distinctive features of GYRA tetraloops. The tetraloop is part of a 27-nt structure that mimics the universal sarcin/ricin loop from Escherichia coli 23S ribosomal RNA in which a GUAA tetraloop replaces the conserved GAGA tetraloop. The adenosines of the GUAA tetraloop form an intermolecular contact that is a commonplace RNA tertiary interaction called an A-minor motif. This is the first structure to reveal in great detail the geometry and hydration of a GUAA tetraloop and an A-minor motif. Comparison of tetraloop structures shows a common backbone geometry for each of the eight possible tetraloop sequences and suggests a common hydration. After backbone atom superposition, equivalent bases from different tetraloops unexpectedly depart from coplanarity by as much as 48 degrees. This variation displaces the functional groups of tetraloops implicated in protein and RNA binding, providing a recognition feature.     

The application of cluster analysis in the intercomparison of loop structures in RNA

We have developed a computational approach for the comparison and classification of RNA loop structures. Hairpin or interior loops identified in atomic resolution RNA structures were intercompared by conformational matching. The root-mean-square deviation (RMSD) values between all pairs of RNA fragments of interest, even if from different molecules, are calculated. Subsequently, cluster analysis is performed on the resulting matrix of RMSD distances using the unweighted pair group method with arithmetic mean (UPGMA). The cluster analysis objectively reveals groups of folds that resemble one another. To demonstrate the utility of the approach, a comprehensive analysis of all the terminal hairpin tetraloops that have been observed in 15 RNA structures that have been determined by X-ray crystallography was undertaken. The method found major clusters corresponding to the well-known GNRA and UNCG types. In addition, two tetraloops with the unusual primary sequence UMAC (M is A or C) were successfully assigned to the GNRA cluster. Larger loop structures were also examined and the clustering results confirmed the occurrence of variations of the GNRA and UNCG tetraloops in these loops and provided a systematic means for locating them. Nineteen examples of larger loops that closely resemble either the GNRA or UNCG tetraloop were found in the large ribosomal RNAs. When the clustering approach was extended to include all structures in the SCOR database, novel relationships were detected including one between the ANYA motif and a less common folding of the GAAA tetraloop sequence.     

Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study

The GNRA (N: any nucleotide; R: purine) tetraloop/receptor interaction is believed to be one of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated tetraloop/receptor complex has not been identified in solution. In the present work, site-directed spin labeling is applied to detect tetraloop/receptor complex formation and estimate the free energy of interaction. For this purpose, the GAAA tetraloop/receptor interaction was chosen as a model system. A method was developed to place nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA tetraloop. Formation of the tetraloop/receptor complex was monitored through changes in the rotational correlation time of the tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA tetraloop forms a complex with an RNA containing the 11-nucleotide GAAA tetraloop receptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 mM MgCl2, Kd = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategy presented here should have broad utility in quantifying weak interactions that would otherwise be undetectable, for both nucleic acids and nucleic acid-protein complexes.     

Unusual nucleotide conformations in GNRA and UNCG type tetraloop hairpins: evidence from Raman markers assignments.

High resolution NMR data on UNCG and GNRA tetraloops (where N is any of the four nucleotides and R is a purine) have shown that they contain ribonucleosides with unusual 2'-endo/anti and 3'-endo/syn conformations, in addition to the 3'-endo/anti ones which are regularly encountered in RNA chains. In the current study, Raman spectroscopy has been used to probe these nucleoside conformations and follow the order (hairpin) to disorder (random chain) structural transitions in aqueous phase in the 5-80 degreesC temperature range. Spectral evolution of GCAA and GAAA tetraloops, as formed in very short hairpins with only three G.C base pairs in their stems (T m >60 degreesC), are reported and compared with those previously published on UUCG and UACG tetraloops, for which the syn orientation of the terminal guanine as well as the 2'-endo/anti conformation of the third rC residue have been confirmed by means of vibrational marker bands. Raman data obtained as a function of temperature show that the first uracil in the UUCG tetraloop is stacked and the two middle residues (rU and rC) are in the 2'-endo/anti conformation, in agreement with the previously published NMR results. As far as the new data concerning the GNRA type tetraloops are concerned, they lead us to conclude that: (i) in both cases (GCAA and GAAA tetraloops) the adenine bases are stacked; (ii) the second rC residue in the GCAA tetraloop has a 3'-endo/anti conformation; (iii) the sugar pucker associated with the third rA residue in both tetraloops possibly undergoes a 3'-endo/2'-endo interconversion as predicted by NMR results; (iv) the stem adopts a regular A-form structure; (v) all other nucleosides of these two GNRA tetraloops possess the usual 3'-endo/anti conformation.     

A novel family of RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNase III.

RNases III are a family of double-stranded RNA (dsRNA) endoribonucleases involved in the processing and decay of a large number of cellular RNAs as well as in RNA interference. The dsRNA substrates of Saccharomyces cerevisiae RNase III (Rnt1p) are capped by tetraloops with the consensus sequence AGNN, which act as the primary docking site for the RNase. We have solved the solution structures of two RNA hairpins capped by AGNN tetraloops, AGAA and AGUU, using NMR spectroscopy. Both tetraloops have the same overall structure, in which the backbone turn occurs on the 3' side of the syn G residue in the loop, with the first A and G in a 5' stack and the last two residues in a 3' stack. A non-bridging phosphate oxygen and the universal G which are essential for Rnt1p binding are strongly exposed. The compared biochemical and structural analysis of various tetraloop sequences defines a novel family of RNA tetraloop fold with the consensus (U/A)GNN and implicates this conserved structure as the primary determinant for specific recognition of Rnt1p substrates.     

RNA tetraloops as minimalist substrates for aminoacylation.

Previous work established that seven-base-pair hairpin microhelices with sequences based on the acceptor stems of alanine, glycine, methionine, and histidine tRNAs can be aminoacylated specifically with their cognate amino acids. To obtain "minimalist" substrates with fewer base pairs, we took advantage of the high thermodynamic stability of RNA tetraloop motifs that are found in ribosomal RNAs. We show here that rationally designed RNA tetraloops with as few as four base pairs are substrates for aminoacylation. Major nucleotide determinants for recognition by the class II synthetases were incorporated into each of the respective tetraloop substrates, resulting in specific aminoacylation by the alanine, glycine, and histidine tRNA synthetases. An analysis of the kinetics of aminoacylation shows that, for the alanine system, the majority of the transition-state stabilization provided by the synthetase-tRNA interaction is reproduced by the interaction of the synthetase with nucleotides in its minimalist tetraloop substrate. In an extension of this work, we also observed specific aminoacylation with the class I methionine tRNA synthetase of RNA tetraloops based on sequences in the acceptor stem of methionine tRNA. Thus, the results demonstrate four different examples where specific aminoacylation is directed by sequences/structures contained in less than half of a turn of an RNA helix.     

Structure of an AAGU tetraloop and its contribution to substrate selection by yeast RNase III

RNase III enzymes are a highly conserved family of proteins that specifically cleave double-stranded RNA (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. In yeast Rnt1p, a dsRNA-binding domain (dsRBD) recognizes its substrate by interacting with stems capped with conserved AGNN tetraloops. The enzyme uses the tetraloop to cut 14nt to 16nt away into the stem in a ruler-like mechanism. The solution structure of Rnt1p dsRBD complexed to one of its small nucleolar (sno) RNA substrate revealed non-sequence-specific contacts with the sugar-phosphate backbone in the minor groove of the AGNN fold and the two non-conserved tetraloop nucleotides. Recently, a new form of Rnt1p substrates lacking the conserved AGNN sequence but instead harboring an AAGU tetraloop was found at the 5' end of snoRNA 48 precursor. Here, we report the solution structure of this hairpin capped with an AAGU tetraloop. Some of the stacking interactions and the position of the turn in the sugar-phosphate backbone are similar to the one observed in the AGNN loop structure; however, the AAGU sequence adopts a different conformation. The most striking difference was found at the 3' end of the loop where Rnt1p interacts with AGNN substrates. The last nucleotide is extruded from the AAGU tetraloop structure in contrast to the compact AGNN fold. The AAGU hairpin structure suggests that Rnt1p recognizes substrates with different tetraloop structures, indicating that the structural repertoire specifically recognized by Rnt1p is larger than previously anticipated.     

Structure of a yeast RNase III dsRBD complex with a noncanonical RNA substrate provides new insights into binding specificity of dsRBDs

dsRBDs often bind dsRNAs with some specificity, yet the basis for this is poorly understood. Rnt1p, the major RNase III in Saccharomyces cerevisiae, cleaves RNA substrates containing hairpins capped by A/uGNN tetraloops, using its dsRBD to recognize a conserved tetraloop fold. However, the identification of a Rnt1p substrate with an AAGU tetraloop raised the question of whether Rnt1p binds to this noncanonical substrate differently than to A/uGNN tetraloops. The solution structure of Rnt1p dsRBD bound to an AAGU-capped hairpin reveals that the tetraloop undergoes a structural rearrangement upon binding to Rnt1p dsRBD to adopt a backbone conformation that is essentially the same as the AGAA tetraloop, and indicates that a conserved recognition mode is used for all Rnt1p substrates. Comparison of free and RNA-bound Rnt1p dsRBD reveals that tetraloop-specific binding requires a conformational change in helix α1. Our findings provide a unified model of binding site selection by this dsRBD.     

The GANC tetraloop: a novel motif in the group IIC intron structure

Tetraloops are a common building block for RNA tertiary structure, and most tetraloops fall into one of three well-characterized classes: GNRA, UNCG, and CUYG. Here, we present the sequence and structure of a fourth highly conserved class of tetraloop that occurs only within the ζ-ζ′ interaction of group IIC introns. This GANC tetraloop was identified, along with an unusual cognate receptor, in the crystal structure of the group IIC intron and through phylogenetic analysis of intron RNA sequence alignments. Unlike conventional tetraloop-receptor interactions, which are stabilized by extensive hydrogen-bonding interactions, the GANC-receptor interaction is limited to a single base stack between the conserved adenosine of the tetraloop and a single purine of the receptor, which consists of a one- to three-nucleotide bulge and does not contain an A-platform. Unlike GNRA tetraloops, the GANC tetraloop forms a sharp angle relative to the adjacent helix, bending by approximately 45° toward the major groove side of the helix. These structural attributes allow GANC tetraloops to fit precisely within the group IIC intron core, thereby demonstrating that structural motifs can adapt to function in a specific niche.     

The GAAA tetraloop-receptor interaction contributes differentially to folding thermodynamics and kinetics for the P4-P6 RNA domain

Tetraloops with the generic sequence GNRA are commonly found in RNA secondary structure, and interactions of such tetraloops with "receptors" elsewhere in RNA play important roles in RNA structure and folding. However, the contributions of tetraloop-receptor interactions specifically to the kinetics of RNA tertiary folding, rather than the thermodynamics of maintaining tertiary structure once folded, have not been reported. Here we investigate the role of the key GAAA tetraloop-receptor motif in folding of the P4-P6 domain of the Tetrahymena group I intron RNA. Insertions of one or more nucleotides into the tetraloop significantly disrupt the thermodynamics of tertiary folding; single-nucleotide insertions shift the folding free energy by 2-4 kcal/mol (DeltaDeltaG(o)'). The folding kinetics of several modified P4-P6 domains were determined by stopped-flow fluorescence spectroscopy, using an internally incorporated pyrene residue as the chromophore. In contrast to the thermodynamic results, the kinetics of Mg(2+)-induced folding were barely affected by the tetraloop modifications, with a DeltaDeltaG(++) of 0.2-0.4 kcal/mol and a Phi value (ratio of the kinetic and thermodynamic contributions) of <0.1. These data indicate an early transition state for folding of P4-P6 with respect to forming the tetraloop-receptor contact, consistent with previous results for modifications elsewhere in P4-P6. We conclude that the GAAA tetraloop-receptor motif contributes little to the stabilization of the transition state for Mg(2+)-induced P4-P6 folding. Rather, the tetraloop-receptor motif acts to clamp the RNA once folding has occurred. This is the first report to correlate the kinetic and thermodynamic contributions of an important RNA tertiary motif, the GNRA tetraloop-receptor. The results are related to possible models for the Mg(2+)-induced folding of the P4-P6 RNA, including a model invoking rapid nonspecific electrostatic collapse.     

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing.

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A-A or A-C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5' side of the loop, exposing their Watson-Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage.     

Solution structure of Cobalt(III)hexammine complexed to the GAAA tetraloop, and metal-ion binding to G.A mismatches

The solution structure of a 22 nt RNA hairpin and its complex with Co(NH(3))(6)(3+) bound to the GAAA tetraloop has been determined by NMR spectroscopy. Co(NH(3))(6)(3+) has a similar geometry to Mg(H(2)O)(6)(2+) and can be used as a probe for binding sites of completely solvated magnesium ions. The hairpin contains tandem G.A mismatches, similar to the P5abc region of a group I intron, and is closed by a GAAA tetraloop. The tandem G.A mismatches are imino hydrogen bonded in contrast with the sheared G.A mismatches found in a different context in the crystal structure of the P4-P6 domain. Chemical shift changes of the imino protons upon titration of the RNA hairpin with Mg(2+) and with Co(NH(3))(6)(3+) were used to identify ion-binding sites. Paramagnetic resonance broadening upon titration with Mn(2+) was also used. The titration curves gave dissociation binding constants for the magnesium ions in the millimolar range, similar to the binding in the major groove of RNA at tandem G.U base-pairs. Although the largest chemical shift change occurred at an imino proton of one of the G.A base-pairs, no nuclear Overhauser enhancement cross-peaks between the cobalt ligand and neighboring RNA protons were seen, presumably due to the high mobility of the Co(NH(3))(6)(3+) at this site. Nuclear Overhauser enhancement cross-peaks between Co(NH(3))(6)(3+) and the GAAA tetraloop were observed, which allowed the determination of the structure of the tetraloop binding site. The Co(NH(3))(6)(3+) is bound in the major groove of the GAAA tetraloop with hydrogen bonds to guanine base N7 and to phosphate oxygen atoms of the tetraloop.     

UNAC tetraloops: to what extent do they mimic GNRA tetraloops?

The structures of four small RNAs each containing a different version of the UNAC loop were determined in solution using NMR spectroscopy and restrained molecular dynamics. The UMAC tetraloops (where M is A or C) exhibited a typical GNRA fold including at least one hydrogen bond between the first U and fourth C. In contrast, UGAC and UUAC tetraloops have a different orientation of the first and fourth residues, such that they do not closely mimic the GNRA fold. Although the UMAC tetraloops are excellent structural mimics of the GNRA tetraloop backbone, sequence comparisons typically do not reveal co‐variation between the two loop types. The limited covariation is attributed to differences in the location of potential hydrogen bond donors and acceptors as a result of the replacement of the terminal A of GNRA with C in the UMAC version. Thus, UMAC loops do not readily form the common GNRA tetraloop‐receptor interaction. The loop at positions 863‐866 in E. coli 16S ribosomal RNA appears to be a major exception. However, in this case the GNRA loop does not in fact engage in the usual base to backbone tertiary interactions. In summary, UMAC loops are not just an alternative sequence version of the GNRA loop family, but instead they expand the types of interactions, or lack thereof, that are possible. From a synthetic biology perspective their inclusion in an artificial RNA may allow the establishment of a stable loop structure while minimizing unwanted long range interactions or permitting alternative long‐range interactions. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 617–628, 2012.     

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] < [Ca(2+)], [Mg(2+)], [Mn(2+)] < [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.     

Increase in hybridization rates with oligodeoxyribonucleotides containing locked nucleic acids

Locked nucleic acids (LNAs) incorporated into either stable single stranded oligonucleotides containing tetraloops or their complements have been found to increase second order hybridization rate constants by an order of magnitude compared to the all-DNA hybridization rate constants. Model sequences composed of 20 bases in length that can form hairpins due to a stable GAAA tetraloop were used where LNAs were substituted for the nucleotides in the loop, stem, or end regions of the strand and in the complementary strand. Substitution of the LNAs to the loop predictably raised the melting temperatures of the duplex however, the hybridization rates between the tetraloop and the complementary sequence also increased. In contrast, when LNAs were substituted in the stem, the hybridization rate decreased implying the formation of a more stable hairpin. Substitution of LNAs into the end region of the sequence had little effect on the hybridization rate constants although melting temperatures still showed a predictable increase. Rates also increased when LNAs were substituted into complementary strands of DNA tetraloops. The increase in hybridization rate constant is being attributed to changes in the structure of the stable single strands.     

Characterization of an RNA receptor motif that recognizes a GCGA tetraloop

Tertiary interactions between a new RNA motif and RNA tetraloops were analyzed to determine whether this new motif shows preference for a GCGA tetraloop. In the structural context of a ligase ribozyme, this motif discriminated GCGA loop from 3 other tetraloops. The affinity between the GCGA loop and its receptor is strong enough to carry out the ribozyme activity.