Tetraloop-receptor Interactions Research Articles

Exploring the energetic and conformational properties of the sequence space connecting naturally occurring RNA tetraloop receptor motifs.

Folded RNAs contain tertiary contact motifs whose structures and energetics are conserved across different RNAs. The transferable properties of RNA motifs simplify the RNA folding problem, but measuring energetic and conformational properties of many motifs remains a challenge. Here, we use a high-throughput thermodynamic approach to investigate how sequence changes alter the binding properties of naturally-occurring motifs, the GAAA tetraloop•tetraloop receptor (TLR) interactions. We measured the binding energies and conformational preferences of TLR sequences that span mutational pathways from the canonical 11ntR to two other natural TLRs, the IC3R and Vc2R. While the IC3R and Vc2R share highly similar energetic and conformational properties, the landscapes that map the sequence changes for their conversion from the 11ntR to changes in these properties differ dramatically. Differences in the energetic landscapes stem from the mutations needed to convert the 11ntR to the IC3R and Vc2R rather than a difference in the intrinsic energetic architectures of these TLRs. The conformational landscapes feature several non-native TLR variants with conformational preferences that differ from both the initial and final TLRs; these species represent potential branching points along the multidimensional sequence space to sequences with greater fitness in other RNA contexts with alternative conformational preferences. Our high-throughput, quantitative approach reveals the complex nature of sequence-fitness landscapes and leads to models for their molecular origins. Systematic and quantitative molecular approaches provide critical insights into understanding the evolution of natural RNAs as they traverse complex landscapes in response to selective pressures.

Studying metal ion binding properties of a three-way junction RNA by heteronuclear NMR.

Self-splicing group II introns are highly structured RNA molecules, containing a characteristic secondary and catalytically active tertiary structure, which is formed only in the presence of Mg(II). Mg(II) initiates the first folding step governed by the κζ element within domain 1 (D1κζ). We recently solved the NMR structure of D1κζ derived from the mitochondrial group II intron ribozyme Sc.ai5γ and demonstrated that Mg(II) is essential for its stabilization. Here, we performed a detailed multinuclear NMR study of metal ion interactions with D1κζ, using Cd(II) and cobalt(III)hexammine to probe inner- and outer-sphere coordination of Mg(II) and thus to better characterize its binding sites. Accordingly, we mapped (1)H, (15)N, (13)C, and (31)P spectral changes upon addition of different amounts of the metal ions. Our NMR data reveal a Cd(II)-assisted macrochelate formation at the 5'-end triphosphate, a preferential Cd(II) binding to guanines in a helical context, an electrostatic interaction in the ζ tetraloop receptor and various metal ion interactions in the GAAA tetraloop and κ element. These results together with our recently published data on Mg(II) interaction provide a much better understanding of Mg(II) binding to D1κζ, and reveal how intricate and complex metal ion interactions can be.

Crystal structure of a eukaryotic group II intron lariat.

The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5′ splice site selection and consists of a 2′-5′ phosphodiester bond between a bulged adenosine and the 5′ end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η-η’ and π-π’, place domain VI in the core to position the lariat bond in the post-catalytic state. Based on structural and biochemical data, we propose that π-π’ is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η’ serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5′ end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

An RNA folding motif: GNRA tetraloop–receptor interactions

Nearly two decades after Westhof and Michel first proposed that RNA tetraloops may interact with distal helices, tetraloop–receptor interactions have been recognized as ubiquitous elements of RNA tertiary structure. The unique architecture of GNRA tetraloops (N=any nucleotide, R=purine) enables interaction with a variety of receptors, e.g., helical minor grooves and asymmetric internal loops. The most common example of the latter is the GAAA tetraloop–11 nt tetraloop receptor motif. Biophysical characterization of this motif provided evidence for the modularity of RNA structure, with applications spanning improved crystallization methods to RNA tectonics. In this review, we identify and compare types of GNRA tetraloop–receptor interactions. Then we explore the abundance of structural, kinetic, and thermodynamic information on the frequently occurring and most widely studied GAAA tetraloop–11 nt receptor motif. Studies of this interaction have revealed powerful paradigms for structural assembly of RNA, as well as providing new insights into the roles of cations, transition states and protein chaperones in RNA folding pathways. However, further research will clearly be necessary to characterize other tetraloop–receptor and long-range tertiary binding interactions in detail – an important milestone in the quantitative prediction of free energy landscapes for RNA folding.

Structural Variation and Uniformity among Tetraloop-Receptor Interactions and Other Loop-Helix Interactions in RNA Crystal Structures

Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the “standard” GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.

The Molecular Interactions That Stabilize RNA Tertiary Structure: RNA Motifs, Patterns, and Networks

RNA molecules adopt specific three-dimensional structures critical to their function. Many essential metabolic processes, including protein synthesis and RNA splicing, are carried out by RNA molecules with elaborate tertiary structures (e.g. 3QIQ, right). Indeed, the ribosome and self-splicing introns are complex RNA machines. But even the coding regions in messenger RNAs and viral RNAs are flanked by highly structured untranslated regions, which provide regulatory information necessary for gene expression. RNA tertiary structure is defined as the three-dimensional arrangement of RNA building blocks, which include helical duplexes, triple-stranded structures, and other components that are held together through connections collectively termed RNA tertiary interactions. The structural diversity of these interactions is now a subject of intense investigation, involving the techniques of NMR, X-ray crystallography, chemical genetics, and phylogenetic analysis. At the same time, many investigators are using biophysical techniques to elucidate the driving forces for tertiary structure formation and the mechanisms for its stabilization. RNA tertiary folding is promoted by maximization of base stacking, much like the hydrophobic effect that drives protein folding. RNA folding also requires electrostatic stabilization, both through charge screening and site binding of metals, and it is enhanced by desolvation of the phosphate backbone. In this Account, we provide an overview of the features that specify and stabilize RNA tertiary structure. A major determinant for overall tertiary RNA architecture is local conformation in secondary-structure junctions, which are regions from which two or more duplexes project. At junctions and other structures, such as pseudoknots and kissing loops, adjacent helices stack on one another, and these coaxial stacks play a major role in dictating the overall architectural form of an RNA molecule. In addition to RNA junction topology, a second determinant for RNA tertiary structure is the formation of sequence-specific interactions. Networks of triple helices, tetraloop-receptor interactions, and other sequence-specific contacts establish the framework for the overall tertiary fold. The third determinant of tertiary structure is the formation of stabilizing stacking and backbone interactions, and many are not sequence specific. For example, ribose zippers allow 2'-hydroxyl groups on different RNA strands to form networks of interdigitated hydrogen bonds, serving to seal strands together and thereby stabilize adjacent substructures. These motifs often require monovalent and divalent cations, which can interact diffusely or through chelation to specific RNA functional groups. As we learn more about the components of RNA tertiary structure, we will be able to predict the structures of RNA molecules from their sequences, thereby obtaining key information about biological function. Understanding and predicting RNA structure is particularly important given the recent discovery that although most of our genome is transcribed into RNA molecules, few of them have a known function. The prevalence of RNA viruses and pathogens with RNA genomes makes RNA drug discovery an active area of research. Finally, knowledge of RNA structure will facilitate the engineering of supramolecular RNA structures, which can be used as nanomechanical components for new materials. But all of this promise depends on a better understanding of the RNA parts list, and how the pieces fit together.

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.

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA.

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.

Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis.

Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity. By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2'-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) that are most important for composing the catalytic core of the intron. The majority of interference effects occur in clusters located within the two catalytically essential Domains 1 and 5 (D1 and D5). Collectively, the NAIM results indicate that key tetraloop-receptor interactions display a specific chemical signature, that the epsilon-epsilon' interaction includes an elaborate array of additional features and that one of the most important core structures is an uncharacterized three-way junction in D1. By combining NAIM with site-directed mutagenesis, a new tertiary interaction, kappa-kappa', was identified between this region and the most catalytically important section of D5, adjacent to the AGC triad in stem 1. Together with the known zeta-zeta' interaction, kappa-kappa' anchors D5 firmly into the D1 scaffold, thereby presenting chemically essential D5 functionalities for participation in catalysis.