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Published January 2016 | Supplemental Material + Published
Journal Article Open

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

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 (preQ_1)-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 preQ_1 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.

Additional Information

© 2016 Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Received 18 September 2015; Accepted 21 October 2015; Published 19 January 2016. We thank J. Widom and K. Suddala for careful reading of the manuscript, and G. Garcia for the generous gift of preQ1. We thank the Single Molecule Analysis in Real-Time (SMART) Center of the University of Michigan, seeded by NSF MRI-R2-ID award DBI-0959823 to N.G.W., as well as J.D. Hoff for performing the Fluorescence correlation spectroscopy measurements. This research was funded by NIH grants GM062357, GM098023 and a sub-contract on GM063162 (PI: J. E. Wedekind) to N.G.W. Author contributions: A.J.R. and N.G.W. conceived of the project. A.J.R. designed, conducted and analysed the SiM-KARTS experiments. M.R.B. wrote and implemented the scripts for burst analysis. P.E.L. designed, conducted and analysed the in vitro translation experiments and SiM-KARTS experiments performed in the presence of blocking strand. M.R.B. and P.E.L. wrote and implemented scripts for burst analysis. All authors were involved in interpreting and discussing the results, and participated in writing the paper. The authors declare no competing financial interests.

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Created:
August 20, 2023
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