Anthony M. Mustoe

Functional complexity and regulation through RNA dynamics.

Changes to the conformation of coding and non-coding RNAs form the basis of elements of genetic regulation and provide an important source of complexity, which drives many of the fundamental processes of life. Although the structure of RNA is highly flexible, the underlying dynamics of RNA are robust and are limited to transitions between the few conformations that preserve favourable base-pairing and stacking interactions. The mechanisms by which cellular processes harness the intrinsic dynamic behaviour of RNA and use it within functionally productive pathways are complex. The versatile functions and ease by which it is integrated into a wide variety of genetic circuits and biochemical pathways suggests there is a general and fundamental role for RNA dynamics in cellular processes.

Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure

Riboswitches are structural elements in the 5′ untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ1 (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ1 riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at near-physiological Mg2+ concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Gō-model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers′ pre-folded states and their ligand binding affinities.

Topological Constraints: Using RNA Secondary Structure to Model 3D Conformation, Folding Pathways, and Dynamic Adaptation

Accompanying recent advances in determining RNA secondary structure is the growing appreciation for the importance of relatively simple topological constraints, encoded at the secondary structure level, in defining the overall architecture, folding pathways, and dynamic adaptability of RNA. A new view is emerging in which tertiary interactions do not define RNA 3D structure, but rather, help select specific conformers from an already narrow, topologically pre-defined conformational distribution. Studies are providing fundamental insights into the nature of these topological constraints, how they are encoded by the RNA secondary structure, and how they interplay with other interactions, breathing new meaning to RNA secondary structure. New approaches have been developed that take advantage of topological constraints in determining RNA backbone conformation based on secondary structure, and a limited set of other, easily accessible constraints. Topological constraints are also providing a much-needed framework for rationalizing and describing RNA dynamics and structural adaptation. Finally, studies suggest that topological constraints may play important roles in steering RNA folding pathways. Here, we review recent advances in our understanding of topological constraints encoded by the RNA secondary structure.

Coarse grained models reveal essential contributions of topological constraints to the conformational free energy of RNA bulges.

Recent studies have shown that simple stereochemical constraints encoded at the RNA secondary structure level significantly restrict the orientation of RNA helices across two-way junctions and yield physically reasonable distributions of RNA 3D conformations. Here we develop a new coarse-grain model, TOPRNA, that is optimized for exploring detailed aspects of these topological constraints in complex RNA systems. Unlike prior models, TOPRNA effectively treats RNAs as collections of semirigid helices linked by freely rotatable single strands, allowing us to isolate the effects of secondary structure connectivity and sterics on 3D structure. Simulations of bulge junctions show that TOPRNA captures new aspects of topological constraints, including variations arising from deviations in local A-form structure, translational displacements of the helices, and stereochemical constraints imposed by bulge-linker nucleotides. Notably, these aspects of topological constraints define free energy landscapes that coincide with the distribution of bulge conformations in the PDB. Our simulations also quantitatively reproduce NMR RDC measurements made on HIV-1 TAR at low salt concentrations, although not for different TAR mutants or at high salt concentrations. Our results confirm that topological constraints are an important determinant of bulge conformation and dynamics and demonstrate the utility of TOPRNA for studying the topological constraints of complex RNAs.

New insights into the fundamental role of topological constraints as a determinant of two-way junction conformation

Recent studies have shown that topological constraints encoded at the RNA secondary structure level involving basic steric and stereochemical forces can significantly restrict the orientations sampled by helices across two-way RNA junctions. Here, we formulate these topological constraints in greater quantitative detail and use this topological framework to rationalize long-standing but poorly understood observations regarding the basic behavior of RNA two-way junctions. Notably, we show that the asymmetric nature of the A-form helix and the finite length of a bulge provide a physical basis for the experimentally observed directionality and bulge-length amplitude dependence of bulge induced inter-helical bends. We also find that the topologically allowed space can be modulated by variations in sequence, particularly with the addition of non-canonical GU base pairs at the junction, and, surprisingly, by the length of the 5′ and 3′ helices. A survey of two-way RNA junctions in the protein data bank confirms that junction residues have a strong preference to adopt looped-in, non-canonically base-paired conformations, providing a route for extending our bulge-directed framework to internal loop motifs and implying a simplified link between secondary and tertiary structure. Finally, our results uncover a new simple mechanism for coupling junction-induced topological constraints with tertiary interactions.

Topological constraints are major determinants of tRNA tertiary structure and dynamics and provide basis for tertiary folding cooperativity

Recent studies have shown that basic steric and connectivity constraints encoded at the secondary structure level are key determinants of 3D structure and dynamics in simple two-way RNA junctions. However, the role of these topological constraints in higher order RNA junctions remains poorly understood. Here, we use a specialized coarse-grained molecular dynamics model to directly probe the thermodynamic contributions of topological constraints in defining the 3D architecture and dynamics of transfer RNA (tRNA). Topological constraints alone restrict tRNAs allowed conformational space by over an order of magnitude and strongly discriminate against formation of non-native tertiary contacts, providing a sequence independent source of folding specificity. Topological constraints also give rise to long-range correlations between the relative orientation of tRNAs helices, which in turn provides a mechanism for encoding thermodynamic cooperativity between distinct tertiary interactions. These aspects of topological constraints make it such that only several tertiary interactions are needed to confine tRNA to its native global structure and specify functionally important 3D dynamics. We further show that topological constraints are conserved across tRNAs different naturally occurring secondary structures. Taken together, our results emphasize the central role of secondary-structure-encoded topological constraints in defining RNA 3D structure, dynamics and folding.

Efficient in silico exploration of RNA interhelical conformations using Euler angles and WExplore

HIV-1 TAR RNA is a two-helix bulge motif that plays a critical role in HIV viral replication and is an important drug target. However, efforts at designing TAR inhibitors have been challenged by its high degree of structural flexibility, which includes slow large-amplitude reorientations of its helices with respect to one another. Here, we use the recently introduced algorithm WExplore in combination with Euler angles to achieve unprecedented sampling of the TAR conformational ensemble. Our ensemble achieves similar agreement with experimental NMR data when compared with previous TAR computational studies, and is generated at a fraction of the computational cost. It clearly emerges from configuration space network analysis that the intermittent formation of the A22-U40 base pair acts as a reversible switch that enables sampling of interhelical conformations that would otherwise be topologically disallowed. We find that most previously determined ligand-bound structures are found in similar location in the network, and we use a sample-and-select approach to guide the construction of a set of novel conformations which can serve as the basis for future drug development efforts. Collectively, our findings demonstrate the utility of WExplore in combination with suitable order parameters as a method for exploring RNA conformational space.

Noncanonical Secondary Structure Stabilizes Mitochondrial tRNASer(UCN) by Reducing the Entropic Cost of Tertiary Folding

Mammalian mitochondrial tRNA(Ser(UCN)) (mt-tRNA(Ser)) and pyrrolysine tRNA (tRNA(Pyl)) fold to near-canonical three-dimensional structures despite having noncanonical secondary structures with shortened interhelical loops that disrupt the conserved tRNA tertiary interaction network. How these noncanonical tRNAs compensate for their loss of tertiary interactions remains unclear. Furthermore, in human mt-tRNA(Ser), lengthening the variable loop by the 7472insC mutation reduces mt-tRNA(Ser) concentration in vivo through poorly understood mechanisms and is strongly associated with diseases such as deafness and epilepsy. Using simulations of the TOPRNA coarse-grained model, we show that increased topological constraints encoded by the unique secondary structure of wild-type mt-tRNA(Ser) decrease the entropic cost of folding by ∼2.5 kcal/mol compared to canonical tRNA, offsetting its loss of tertiary interactions. Further simulations show that the pathogenic 7472insC mutation disrupts topological constraints and hence destabilizes the mutant mt-tRNA(Ser) by ∼0.6 kcal/mol relative to wild-type. UV melting experiments confirm that insertion mutations lower mt-tRNA(Ser) melting temperature by 6-9 °C and increase the folding free energy by 0.8-1.7 kcal/mol in a largely sequence- and salt-independent manner, in quantitative agreement with our simulation predictions. Our results show that topological constraints provide a quantitative framework for describing key aspects of RNA folding behavior and also provide the first evidence of a pathogenic mutation that is due to disruption of topological constraints.

An RNA tertiary switch by modifying how helices are tethered

A viral tRNA-like structure has evolved a unique strategy to undergo a tertiary structure conformational switch that may help regulate viral regulation.

Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

A requirement for specific RNA folding is that the free-energy landscape discriminate against non-native folds. While tertiary interactions are critical for stabilizing the native fold, they are relatively non-specific, suggesting additional mechanisms contribute to tertiary folding specificity. In this study, we use coarse-grained molecular dynamics simulations to explore how secondary structure shapes the tertiary free-energy landscape of the Azoarcus ribozyme. We show that steric and connectivity constraints posed by secondary structure strongly limit the accessible conformational space of the ribozyme, and that these so-called topological constraints in turn pose strong free-energy penalties on forming different tertiary contacts. Notably, native A-minor and base-triple interactions form with low conformational free energy, while non-native tetraloop/tetraloop–receptor interactions are penalized by high conformational free energies. Topological constraints also give rise to strong cooperativity between distal tertiary interactions, quantitatively matching prior experimental measurements. The specificity of the folding landscape is further enhanced as tertiary contacts place additional constraints on the conformational space, progressively funneling the molecule to the native state. These results indicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary folding landscape, and further emphasize topological constraints as a key force in RNA folding.