The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts.

In response to intracellular signals in Gram--negative bacteria, translational riboswitches--commonly embedded in messenger RNAs (mRNAs)-regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by 'bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation.

Native purification and labeling of RNA for single molecule fluorescence studies.

The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure-function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5' end, sometimes with the aid of protein chaperones. By contrast, in the laboratory RNAs are commonly generated by in vitro transcription or chemical synthesis, followed by purification in a manner that includes the use of high concentrations of urea, heat and UV light (for detection), resulting in the denaturation and subsequent refolding of the entire RNA. Recent studies into the nature of heterogeneous RNA populations resulting from this process have underscored the need for non-denaturing (native) purification methods that maintain the co-transcriptional fold of an RNA. Here, we present protocols for the native purification of an RNA after its in vitro transcription and for fluorophore and biotin labeling methods designed to preserve its native conformation for use in single molecule fluorescence resonance energy transfer (smFRET) inquiries into its structure and function. Finally, we present methods for taking smFRET data and for analyzing them, as well as a description of plausible overall preparation schemes for the plethora of non-coding RNAs.

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 Mg(2+) concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Go-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.