Nathan J. Baird

Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch

The cyclic diguanylate (bis-(3′-5′)-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis

Riboswitches are structured mRNA elements that regulate gene expression upon binding specific cellular metabolites. It is thought that the highly conserved metabolite-binding domains of riboswitches undergo conformational change upon binding their cognate ligands. To investigate the generality of such a mechanism, we employed small-angle X-ray scattering (SAXS). We probed the nature of the global metabolite-induced response of the metabolite-binding domains of four different riboswitches that bind, respectively, thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), lysine, and S-adenosyl methionine (SAM). We find that each RNA is unique in its global structural response to metabolite. Whereas some RNAs exhibit distinct free and bound conformations, others are globally insensitive to the presence of metabolite. Thus, a global conformational change of the metabolite-binding domain is not a requirement for riboswitch function. It is possible that the range of behaviors observed by SAXS, rather than being a biophysical idiosyncrasy, reflects adaptation of riboswitches to the regulatory requirements of their individual genomic context.

Riboswitch function: Flipping the switch or tuning the dimmer?

Riboswitches are structured mRNA elements involved in gene regulation that respond to the intracellular concentration of specific small molecules. Binding of their cognate ligand is thought to elicit a global conformational change of the riboswitch, in addition to modulating the fine structure of the binding site. X-ray crystallography has produced detailed descriptions of the three-dimensional structures of the ligand-bound conformations of several riboswitches. We have employed small-angle X-ray scattering (SAXS) to generate low-resolution reconstructions of the ligand-free states of the ligand-binding domains of riboswitches that respond to thiamine pyrophosphate (TPP), and cyclic diguanylate (c-di-GMP), a bacterial second messenger. Comparison of the SAXS reconstructions with the crystal structures of these two riboswitches demonstrates that the RNAs undergo dramatic ligand-induced global conformational changes. However, this is not an universal feature of riboswitches. SAXS analysis of the solution behavior of several other riboswitch ligand-binding domains demonstrates a broad spectrum of conformational switching behaviors, ranging from the unambiguous switching of the TPP and c-di-GMP riboswitches to complete lack of switching for the flavin mononucleotide (FMN) riboswitch. Moreover, the switching behavior varies between examples of the same riboswitch from different organisms. The range of observed behaviors suggests that in response to the evolutionary need for precise genetic regulation, riboswitches may be tuned to function more as dimmers or rheostats than binary on/off switches.

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops.

The archaeal protein L7Ae and eukaryotic homologs such as L30e and 15.5kD comprise the best characterized family of K-turn-binding proteins. K-turns are an RNA motif comprised of a bulge flanked by canonical and noncanonical helices. They are widespread in cellular RNAs, including bacterial gene-regulatory RNAs such as the c-di-GMP-II, lysine, and SAM-I riboswitches, and the T-box. The existence in bacteria of K-turn-binding proteins of the L7Ae family has not been proven, although two hypothetical proteins, YbxF and YlxQ, have been proposed to be L7Ae homologs based on sequence conservation. Using purified, recombinant proteins, we show that Bacillus subtilis YbxF and YlxQ bind K-turns (K(d) ~270 nM and ~2300 nM, respectively). Crystallographic structure determination demonstrates that both YbxF and YlxQ adopt the same overall fold as L7Ae. Unlike the latter, neither bacterial protein recognizes K-loops, a structural motif that lacks the canonical helix of the K-turn. This property is shared between the bacterial and eukaryal family members. Comparison of our structure of YbxF in complex with the K-turn of the SAM-I riboswitch and previously determined structures of archaeal and eukaryal homologs bound to RNA indicates that L7Ae approaches the K-turn at a unique angle, which results in a considerably larger RNA-protein interface dominated by interactions with the noncanonical helix of the K-turn. Thus, the inability of the bacterial and eukaryal L7Ae homologs to bind K-loops probably results from their reliance on interactions with the canonical helix. The biological functions of YbxF and YlxQ remain to be determined.

Folding of a universal ribozyme: the ribonuclease P RNA

Ribonuclease P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the folding studies of bacterial RNase P RNA.

Discrete Structure of an RNA Folding Intermediate Revealed by Cryo-electron Microscopy

RNA folding occurs via a series of transitions between metastable intermediate states. It is unknown whether folding intermediates are discrete structures folding along defined pathways or heterogeneous ensembles folding along broad landscapes. We use cryo-electron microscopy and single-particle image reconstruction to determine the structure of the major folding intermediate of the specificity domain of a ribonuclease P ribozyme. Our results support the existence of a discrete conformation for this folding intermediate.

Extended structures in RNA folding intermediates are due to nonnative interactions rather than electrostatic repulsion.

RNA folding occurs via a series of transitions between metastable intermediate states for Mg(2+) concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154-residue specificity domain (S-domain) of the Bacillus subtilis RNase P RNA is more extended than its native structure. We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the Escherichia coli RNase P RNA that belong to different classes of P RNA and have distinct native structures. The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering and Langevin dynamics simulations, we show that electrostatics plays only a minor role, whereas specific interactions largely account for the extended nature of the intermediate. Structural contacts in the core, including a nonnative base pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, nonnative interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes.

Rapid RNA-ligand interaction analysis through high-information content conformational and stability landscapes

The structure and biological properties of RNAs are a function of changing cellular conditions, but comprehensive, simultaneous investigation of the effect of multiple interacting environmental variables is not easily achieved. We have developed an efficient, high-throughput method to characterize RNA structure and thermodynamic stability as a function of multiplexed solution conditions using Förster resonance energy transfer (FRET). In a single FRET experiment using conventional quantitative PCR instrumentation, 19,400 conditions of MgCl2, ligand and temperature are analysed to generate detailed empirical conformational and stability landscapes of the cyclic diguanylate (c-di-GMP) riboswitch. The method allows rapid comparison of RNA structure modulation by cognate and non-cognate ligands. Landscape analysis reveals that kanamycin B stabilizes a non-native, idiosyncratic conformation of the riboswitch that inhibits c-di-GMP binding. This demonstrates that allosteric control of folding, rather than direct competition with cognate effectors, is a viable approach for pharmacologically targeting riboswitches and other structured RNA molecules.

Analysis of riboswitch structure and ligand binding using small-angle X-ray scattering (SAXS).

Small-angle X-ray scattering (SAXS) is a powerful tool for examining the global conformation of riboswitches in solution, and how this is modulated by binding of divalent cations and small molecule ligands. SAXS experiments, which typically require only minutes per sample, directly yield two quantities describing the size and shape of the RNA: the radius of gyration (Rg) and the maximum linear dimension (Dmax). Examination of these quantities can reveal if a riboswitch undergoes cation-induced compaction. Comparison of the Rg and Dmax values between samples containing different concentrations of ligand reveals the overall structural response of the riboswitch to ligand. The Kratky plot (a graphical representation that emphasizes the higher-resolution SAXS data) and the P(r) plot or pair-probability distribution (an indirect Fourier transform, or power spectrum of the data) can provide additional evidence of riboswitch conformational changes. Simulation methods have been developed for generating three-dimensional reconstructions consistent with the one-dimensional SAXS data. These low-resolution molecular envelopes can aid in deciphering the relative helical arrangement within the RNA.