Journal
NATURE COMMUNICATIONS
Volume 7, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/ncomms9976
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Funding
- NSF MRI-R2-ID award [DBI-0959823]
- NIH [GM062357, GM098023]
- [GM063162]
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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 RNATransient 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.
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