Chemical probing of RNA and DNA structure is a widely used and highly informative approach for examining nucleic acid structure and for evaluating interactions with protein and small-molecule ligands. Use of capillary electrophoresis to analyze chemical probing experiments yields hundreds of nucleotides of information per experiment and can be performed on automated instruments. Extraction of the information from capillary electrophoresis electropherograms is a computationally intensive multistep analytical process, and no current software provides rapid, automated, and accurate data analysis. To overcome this bottleneck, we developed a platform-independent, user-friendly software package, QuShape, that yields quantitatively accurate nucleotide reactivity information with minimal user supervision. QuShape incorporates newly developed algorithms for signal decay correction, alignment of time-varying signals within and across capillaries and relative to the RNA nucleotide sequence, and signal scaling across channels or experiments. An analysis-by-reference option enables multiple, related experiments to be fully analyzed in minutes. We illustrate the usefulness and robustness of QuShape by analysis of RNA SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) experiments.

The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2′-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2′-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2′-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the E. coli ribosome, monitored the reactivity of 1490 nucleotides in 16S ribosomal RNA, and examined those nucleotides that were hyper-reactive towards SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2′-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3′-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry.

There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand-binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute timescale using SHAPE. The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg2+, the approximate Mg2+ concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg2+ concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg2+ alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations.

RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and non-additive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes.

It was shown decades ago that purified 30S ribosome subunits readily interconvert between "active" and "inactive" conformations in a switch that involves changes in the functionally important neck and decoding regions. However, the physiological significance of this conformational change had remained unknown. In exponentially growing Escherichia coli cells, RNA SHAPE probing revealed that 16S rRNA largely adopts the inactive conformation in stably assembled, mature 30S subunits and the active conformation in translating (70S) ribosomes. Inactive 30S subunits bind mRNA as efficiently as active subunits but initiate translation more slowly. Mutations that inhibited interconversion between states compromised translation in vivo. Binding by the small antibiotic paromomycin induced the inactiveto-active conversion, consistent with a low-energy barrier between the two states. Despite the small energetic barrier between states, but consistent with slow translation initiation and a functional role in vivo, interconversion involved large-scale changes in structure in the neck region that likely propagate across the 30S body via helix 44. These findings suggest the inactive state is a biologically relevant alternate conformation that regulates ribosome function as a conformational switch. orty-five years ago, Zamir, Elson, and their colleagues reported that purified 30S subunits of the ribosome undergo a readily reversible conformational change between "active" and "inactive" states and proposed that this conformational rearrangement might mimic a natural process (1). Noller and coworkers used chemical probing to show that this conformational change occurs in the neck and decoding center regions of the 16S ribosomal RNA (rRNA) and has "the appearance of a reciprocal interconversion between two differently structured states" (2). Recent structural analyses indicate that the protein-free 16S rRNA adopts alternative base-paired conformations in the neck region that are conserved among diverse eubacterial and archeal organisms (3). The ability to sample multiple conformations in this region is also conserved in eukaryotes (4). The original studies on the inactive and active states noted that probing ribosomes in cells might allow the biological roles of these states to be established (1, 2). Here we make use of recent innovations in in-cell RNA SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) probing (5) to interrogate the structure of 16S rRNA in free 30S subunits, in actively translating ribosomes, and in mutant ribosomes in exponentially growing Escherichia coli. ResultsIn Vivo SHAPE Probing of Ribosomal States. We used in vivo SHAPE (5, 6) to probe the RNA structure in exponentially growing E. coli cells and then halted translation by rapidly pouring the cells over ice (7). Experiments were performed with the SHAPE reagent 1M7, which readily enters cells and either reacts with RNA or undergoes inactivation by hydrolysis over ∼2 min. Probing is thus rapid, no explicit quench step is required, and the ex...