A mutate-and-map protocol for inferring base pairs in structured RNA

Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecule's reactivity to different probes is quantified at single-nucleotide resolution and used to constrain structural modeling. This experimental framework has been extensively revisited in the past decade with new strategies for high-throughput read-outs, chemical modification, and rapid data analysis. Recently, we have coupled the technique to high-throughput mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure of not only that nucleotide but also its interaction partner. Carrying out the mutation and mapping for the entire system gives an experimental approximation of the molecules contact map. Here, we give our in-house protocol for this mutate-and-map strategy, based on 96-well capillary electrophoresis, and we provide practical tips on interpreting the data to infer nucleic acid structure.Comment: 22 pages, 5 figure

Understanding the errors of SHAPE-directed RNA structure modeling

Single-nucleotide-resolution chemical mapping for structured RNA is being rapidly advanced by new chemistries, faster readouts, and coupling to computational algorithms. Recent tests have shown that selective 2'-hydroxyl acylation by primer extension (SHAPE) can give near-zero error rates (0-2%) in modeling the helices of RNA secondary structure. Here, we benchmark the method using six molecules for which crystallographic data are available: tRNA(phe) and 5S rRNA from Escherichia coli, the P4-P6 domain of the Tetrahymena group I ribozyme, and ligand-bound domains from riboswitches for adenine, cyclic di-GMP, and glycine. SHAPE-directed modeling of these highly structured RNAs gave an overall false negative rate (FNR) of 17% and a false discovery rate (FDR) of 21%, with at least one helix prediction error in five of the six cases. Extensive variations of data processing, normalization, and modeling parameters did not significantly mitigate modeling errors. Only one varation, filtering out data collected with deoxyinosine triphosphate during primer extension, gave a modest improvement (FNR = 12%, and FDR = 14%). The residual structure modeling errors are explained by the insufficient information content of these RNAs' SHAPE data, as evaluated by a nonparametric bootstrapping analysis. Beyond these benchmark cases, bootstrapping suggests a low level of confidence (<50%) in the majority of helices in a previously proposed SHAPE-directed model for the HIV-1 RNA genome. Thus, SHAPE-directed RNA modeling is not always unambiguous, and helix-by-helix confidence estimates, as described herein, may be critical for interpreting results from this powerful methodology.Comment: Biochemistry, Article ASAP (Aug. 15, 2011

Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg2+ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gswloop) of the guanine-sensing riboswitch and their Mg2+-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gswloop or the increase in temperature for both Gsw and Gswloop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg2+. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding

The K-turn motif in riboswitches and other RNA species

AbstractThe kink turn is a widespread structure motif that introduces a tight bend into the axis of duplex RNA. This generally functions to mediate tertiary interactions, and to serve as a specific protein binding site. K-turns or closely related structures are found in at least seven different riboswitch structures, where they function as key architectural elements that help generate the ligand binding pocket. This article is part of a Special Issue entitled: Riboswitches

Predicting translational diffusion of evolutionary conserved RNA structures by the nucleotide number

Ribonucleic acids are highly conserved essential parts of cellular life. RNA function is determined to a large extent by its hydrodynamic behaviour. The presented study proposes a strategy to predict the hydrodynamic behaviour of RNA single strands on the basis of the polymer size. By atom-level shell-modelling of high-resolution structures, hydrodynamic radius and diffusion coefficient of evolutionary conserved RNA single strands (ssRNA) were calculated. The diffusion coefficients D of 17–174 nucleotides (nt) containing ssRNA depended on the number of nucleotides N with D = 4.56 × 10−10 N−0.39 m2 s−1. The hydrodynamic radius RH depended on N with RH = 5.00 × 10−10 N0.38 m. An average ratio of the radius of gyration and the hydrodynamic radius of 0.98 ± 0.08 was calculated in solution. The empirical law was tested by in solution measured hydrodynamic radii and radii of gyration and was found to be highly consistent with experimental data of evolutionary conserved ssRNA. Furthermore, the hydrodynamic behaviour of several evolutionary unevolved ribonucleic acids could be predicted. Based on atom-level shell-modelling of high-resolution structures and experimental hydrodynamic data, empirical models are proposed, which enable to predict the translational diffusion coefficient and molecular size of short RNA single strands solely on the basis of the polymer size

Attenuation of loop-receptor interactions with pseudoknot formation

RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more ‘G/C-rich’ than their ‘A/U-rich’ natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for ‘A/U-rich’ GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology

A c-di-GMP Effector System Controls Cell Adhesion by Inside-Out Signaling and Surface Protein Cleavage

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit—from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger

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