Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.     

Structural differences within the loop E motif imply alternative mechanisms of viroid processing

Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires extensive rearrangement of the highly base-paired native structure. For Potato spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by the change from a multibranched tetraloop structure to a loop E conformation. Here we present evidence that processing of Citrus viroid III (CVd-III), a member of a related group of viroids that also replicate in the nucleus, may proceed via a distinct pathway. Chemical probing of PSTVd and CVd-III miniRNAs with DMS and CMCT revealed that the loop E motifs of these two viroids have quite different tertiary structures. As shown by temperature gradient gel electrophoresis, the presence of two likely Watson-Crick GC pairs results in a significant overall stabilization of the CVd-III loop E-like motif. Unlike PSTVd, the upper strand of the CVd-III loop E-like motif cannot fold into a GNRA tetraloop, and comparison of suboptimal structures indicates that the initial cleavage event could occur on the 5' side of the only GU wobble pair in a helix involving a nearby pair of inverted repeats. According to our model, rearrangement of 3' sequences into a hairpin stem containing an identical arrangement of GC, GU, and CG base pairs and a second cleavage event is followed by formation of loop E, which serves to align the 5' and 3' termini of the CVd-III monomer prior to ligation. Because ligation would occur within loop E itself, stabilization of this motif may be needed to hold the 5' and 3' termini of CVd-III in position for the host ligase.     

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.     

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.     

RNA simulations: probing hairpin unfolding and the dynamics of a GNRA tetraloop

Simulations of an RNA hairpin containing a GNRA tetraloop were conducted to allow the characterization of its secondary structure formation and dynamics. Ten 10 ns trajectories of the folded hairpin 5'-GGGC[GCAA]GCCU-3' were generated using stochastic dynamics and the GB/SA implicit solvent model at 300 K. Overall, we find the stem to be a very stable subunit of this molecule, whereas multiple loop conformations and transitions between them were observed. These trajectories strongly suggest that extension of the C6 base away from the loop occurs cooperatively with an N-type-->S-type sugar pucker conversion in that residue and that similar pucker transitions are necessary to stabilize other looped-out bases. In addition, a short-lived conformer with an extended fourth loop residue (A8) lacking this stabilizing 2'-endo pucker mode was observed. Results of thermal perturbation at 400 K support this model of loop dynamics. Unfolding trajectories were produced using this same methodology at temperatures of 500 to 700 K. The observed unfolding events display three-state behavior kinetically (including native, globular, and unfolded populations) and, based on these observations, we propose a folding mechanism that consists of three distinct events: (i) collapse of the random unfolded structure and sampling of the globular state; (ii) passage into the folded region of configurational space as stem base-pairs form and gain helicity; and (iii) attainment of proper loop geometry and organization of loop pairing and stacking interactions. These results are considered in the context of current experimental knowledge of this and similar nucleic acid hairpins.     

Structural organization of a viral IRES depends on the integrity of the GNRA motif

Little is known about the tertiary structure of internal ribosome entry site (IRES) elements. The central domain of foot-and-mouth disease (FMDV) IRES, named 3 or I, contains a conserved GNRA motif, essential for IRES activity. We have combined functional analysis with RNA probing to define its structural organization. We have found that a UNCG motif does not functionally substitute the GNRA motif; moreover, binding of synthetic GNRA stem-loops to domain 3 was significantly reduced in RNAs bearing UCCG or GUAG substitutions. The apical region of domain 3 consists of a four-way junction where residues of the GNRA tetraloop are responsible for the organization of the adjacent stem-loops, as deduced from ribonucleases and dimethyl sulfate accessibility. A single A-to-G substitution in the fourth position of this motif led to a strong RNA reorganization, affecting several nucleotides away in the secondary structure of domain 3. The study of mutants bearing UNCG or GUAG tetraloops revealed lack of protection to chemical attack in native RNA at specific nucleotides relative to the parental GUAA, suggesting that the GNRA motif dictates the organization and stability of domain 3. This effect is likely mediated by the interaction with distant residues. Therefore, the GNRA motif plays a crucial role in the organization of IRES structure with important consequences on activity.     

Molecular dynamics study of intrinsic stability in six RNA terminal loop motifs

Single stranded RNA molecules can assume a wide range of tertiary structures beyond the canonical A-form double helix. Certain sequences, termed motifs, are more common than a random distribution would suggest. The existence of such motifs can be rationalized in structural terms. In this study, we have investigated the intrinsic structural stability of RNA terminal loop motifs using multiple MD simulations in explicit water. Representative loops were chosen from the major tetraloop motifs, including also the U-turn motif. Not all loops retain their folded starting structure, but lowering the temperature to 277 K, or adding adjacent base pairs from the stem to which the motif is attached, helps stabilizing the folded loop structure.     

Rules for RNA recognition of GNRA tetraloops deduced by in vitro selection: comparison with in vivo evolution.

Terminal loops with a GNRA consensus sequence are a prominent feature of large self-assembling RNA molecules. In order to investigate tertiary interactions involving GNRA loops, we have devised an in vitro selection system derived from a group I ribozyme. Two selections, destined to isolate RNA sequences that would recognize two of the most widespread loops (GUGA and GAAA), yielded variants of previously identified receptors for those loops, and also some yet unrecognized, high-affinity binders with novel specificities towards members of the GNRA family. By taking advantage of available crystal structures, we have attempted to rationalize these results in terms of RNA-RNA contacts and to expose some of the structural principles that govern GNRA loop-mediated tertiary interactions; the role of loop nucleotide 2 in ensuring specific recognition by receptors is emphasized. More generally, comparison of the products of in vitro and natural selection is shown to provide insights into the mechanisms underlying the in vivo evolution of self-assembling RNA molecules.     

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.     

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.     

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.     

A conserved motif in group IC3 introns is a new class of GNRA receptor

Terminal tetraloops consisting of GNRA sequences are often found in biologically active large RNAs. The loops appear to contribute towards the organization of higher order RNA structures by forming specific tertiary interactions with their receptors. Group IC3 introns which possess a GAAA loop in the L2 region often have a phylogenetically conserved motif in their P8 domains. In this report, we show that this conserved motif stands as a new class of receptor that distinguishes the sequences of GNRA loops less stringently than previously known receptors. The motif can functionally substitute an 11 nt motif receptor in the Tetrahymena ribozyme. Its structural and functional similarity to one class of synthetic receptors obtained from in vitro selection is observed.     

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures.     

Clustering RNA structural motifs in ribosomal RNAs using secondary structural alignment

RNA structural motifs are the building blocks of the complex RNA architecture. Identification of non-coding RNA structural motifs is a critical step towards understanding of their structures and functionalities. In this article, we present a clustering approach for de novo RNA structural motif identification. We applied our approach on a data set containing 5S, 16S and 23S rRNAs and rediscovered many known motifs including GNRA tetraloop, kink-turn, C-loop, sarcin-ricin, reverse kink-turn, hook-turn, E-loop and tandem-sheared motifs, with higher accuracy than the state-of-the-art clustering method. We also identified a number of potential novel instances of GNRA tetraloop, kink-turn, sarcin-ricin and tandem-sheared motifs. More importantly, several novel structural motif families have been revealed by our clustering analysis. We identified a highly asymmetric bulge loop motif that resembles the rope sling. We also found an internal loop motif that can significantly increase the twist of the helix. Finally, we discovered a subfamily of hexaloop motif, which has significantly different geometry comparing to the currently known hexaloop motif. Our discoveries presented in this article have largely increased current knowledge of RNA structural motifs.     

Achieving specificity in selected and wild-type N peptide-RNA complexes: the importance of discrimination against noncognate RNA targets

The boxB RNA pentaloops from the P22 and lambda phages each adopt a GNRA tetraloop fold upon binding their cognate arginine-rich N peptides. The third loop base in P22 boxB (3-out) and the fourth in lambda boxB (4-out) are excluded to accommodate this structure. Previously, we selected a pool of lambda N sequences with random amino acids at loop contacting positions 13-22 for binding to either of these two GNRA-folded pentaloops or a canonical GNRA tetraloop and isolated a class of peptides with a new conserved arginine (R15). Here, we characterize the binding of lambda N and these R15 peptides using fluorescent titrations with 2-aminopurine labeled versions of the three GNRA-folded loops and circular dichroism spectrometry. All peptides preferentially bind the lambda boxB RNA loop. lambda N and R15 peptide specificity against the P22 loop arises from the cost of rearranging its loop into the 4-out GNRA structure. Modeling indicates that the interaction of R8 with an additional loop phosphate in the 4-out GNRA pentaloop selectively stabilizes this complex relative to the tetraloop. R15 peptides gain additional discrimination against the tetraloop because their arginine also preferentially interacts with the 4-out GNRA pentaloop phosphate backbone, whereas K14 and W18 of lambda N contribute equal affinity when binding the tetraloop. Nonspecific electrostatic interactions by basic residues near the C-termini of these peptides create significantly steeper salt dependencies in association constants for noncognate loops, aiding discrimination at high salt concentrations. Our results emphasize the importance of considering specificity against noncognate as well as nonspecific targets in the combinatorial and rational design of biopolymers capable of macromolecular recognition.     

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 a SRP19 binding domain in human SRP RNA

Assembly of the human signal recognition particle (SRP) requires SRP19 protein to bind to helices 6 and 8 of SRP RNA. In the present study, structure of a 29-mer RNA composing the SRP19 binding site in helix 6 was determined by NMR spectroscopy. The two A:C mismatches were continuously stacked to each other and formed wobble type A:C base pairs. The GGAG tetraloop in helix 6 was found to adopt a similar conformation to that of GNRA tetraloop, suggesting that these tetraloops are included in an extensive new motif GNRR. Compared with the crystal structure of helix 6 in complex with SRP19 determined previously, the GGAG tetraloop in the complex was found to adopt a similar conformation to the free form, although the loop structure becomes more open upon SRP19 binding. Thus, SRP19 is thought to recognize the overall fold of the GGAG loop.     

Structural resiliency of an EGF-like subdomain bound to its target protein, thrombin.

The thrombin-bound structures of native peptide fragments from the fifth EGF-like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF-like structure of an antiparallel beta-sheet, a novel structural motif observed for a bound peptide in protein-peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF-like domains, are unfavorably exposed in the complex of thrombin with the EGF-like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique beta-sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side-chain interactions within a specific environment may dictate the folding of bound peptides in protein-peptide complexes.     

Three-dimensional motifs from the SCOR, structural classification of RNA database: extruded strands, base triples, tetraloops and U-turns

Release 2.0.1 of the Structural Classification of RNA (SCOR) database, http://scor.lbl.gov, contains a classification of the internal and hairpin loops in a comprehensive collection of 497 NMR and X-ray RNA structures. This report discusses findings of the classification that have not been reported previously. The SCOR database contains multiple examples of a newly described RNA motif, the extruded helical single strand. Internal loop base triples are classified in SCOR according to their three-dimensional context. These internal loop triples contain several examples of a frequently found motif, the minor groove AGC triple. SCOR also presents the predominant and alternate conformations of hairpin loops, as shown in the most well represented tetraloops, with consensus sequences GNRA, UNCG and ANYA. The ubiquity of the GNRA hairpin turn motif is illustrated by its presence in complex internal loops.