Abstract
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.
Highlights
In response to intracellular signals in Gram-negative bacteria, translational riboswitches— commonly embedded in messenger RNAs—regulate gene expression through inhibition of translation initiation
Our results suggest that the T. tengcongensis (Tte) messenger RNAs (mRNAs) with embedded preQ1 riboswitch transitions between two distinguishable equilibrium conformational states: a bursting state conformation with an exposed SD sequence that is available for frequent binding of the anti-SD sequence and a non-bursting conformation with a less accessible SD sequence
It is generally thought that translational riboswitches achieve gene regulation through a ligand-mediated conformational change in the aptamer domain that is transduced into the downstream expression platform to actuate an ON/ OFF switch in gene expression[1,2,3,4,5]
Summary
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. Riboswitches are noncoding structural elements most commonly embedded in the 50 untranslated region (UTR) of bacterial messenger RNAs (mRNAs) that regulate the expression of a downstream gene through the binding of an intracellular signal[1,2,3,4,5]. In the absence of ligand, this helix is only transiently closed[19], presumably revealing the SD sequence in its entirety as part of the downstream expression platform to bind the anti-SD sequence of the 16S ribosomal RNA (rRNA, Fig. 1a,b) These and many similar studies[21,22] have provided valuable insight into the conformational sampling of the preQ1 riboswitch as well as other translational riboswitches as a function of ligand concentration. We anticipate that SiM-KARTS will find broad application in probing dynamic RNA structural elements at the single-molecule level
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