Probing Transient Riboswitch Structures via Single Molecule Accessibility Analysis

Abstract

Riboswitches are a class of RNA motifs in the untranslated regions of bacterial messenger RNAs (mRNAs) that can adopt different conformations to regulate gene expression. The binding of specific small molecule or ion ligands, or other RNAs, influences the conformation the riboswitch adopts. Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) offers an approach for probing this structural isomerization, or conformational switching, at the level of single mRNA molecules. SiM-KARTS utilizes fluorescently labeled, short, sequence-complementary DNA or RNA oligonucleotide probes that transiently access a specific RNA conformation over another. Binding and dissociation to a surface-immobilized target RNA of arbitrary length are monitored by Total Internal Reflection Fluorescence Microscopy (TIRFM) and quantitatively analyzed, via spike train and burst detection, to elucidate the rate constants of isomerization, revealing mechanistic insights into riboswitching.

Keywords: Bacterial gene regulation; Conformational dynamics; RNA folding; Riboswitch; Single molecule fluorescence microscopy.

Fig. 1

SiM-KARTS measurements of an mRNA with embedded preQ₁ riboswitch. (A) The preQ₁ riboswitch containing mRNA. The transcript is immobilized onto the microscope slide after hybridization of the biotinylated capture probe (CP) to the corresponding recognition site on the target. The distally hybridized localization probe (LP) allows for the localization of single immobilized mRNA molecules. Transiently binding fluorescence probe (FP, here against the Shine-Dalgarno (SD) sequence of the TTE1564 protein) senses structural changes of the riboswitch. (B) Experimental schematic of the SiM-KARTS assay using a prism-based TIRFM setup. After the RNA complex is immobilized onto a microscope slide via streptavidin and biotinylated BSA (biotin-BSA, not shown for simplicity), it is illuminated with a 532 nm wavelength laser (exciting the LP) to identify the RNA complex. A red laser (638 nm) is used to excite the FP, whose repeated binding to the Shine-Dalgarno (SD) sequence results in spikes of fluorescence. In the presence of preQ₁, the accessibility of the binding site of the FP changes.

Fig. 2

Example single molecule fluorescence trace (blue), idealized here by intensity thresholding (orange).

Fig. 3

Extraction of burst behavior of a labeled SiM-KARTS probe binding and dissociating via spike train analysis. The blue line represents simulated data. Boxes indicate the time the RNA is in a conformation where the binding site is readily accessible (blue; burst) or inaccessible (pink; non-burst).

Fig. 4

Normalized histogram representing the distribution of ISIs during bursts (when the binding site is accessible; in blue) and non-burst (when the binding site is not readily accessible; in red). Inset: Cumulative histogram representation of the same data.