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

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