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

Nature Communications
Arlie J RinaldiNils G Walter

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.

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Citations

Apr 5, 2017·Annual Review of Biophysics·Christopher P Jones, Adrian R Ferré-D'Amaré
Jul 14, 2017·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Sha GongWenbing Zhang
Nov 18, 2017·International Journal of Molecular Sciences·Sha GongWenbing Zhang
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Oct 21, 2016·Nature Protocols·Jinglin FuHao Yan
Dec 14, 2019·Nucleic Acids Research·Paul E LundNils G Walter
Oct 30, 2019·The Journal of Biological Chemistry·Debapratim Dutta, Joseph E Wedekind
Sep 22, 2019·Nature Communications·Krishna C SuddalaNils G Walter
Dec 28, 2016·PLoS Computational Biology·Wonseok HwangChangbong Hyeon
Jul 6, 2017·Scientific Reports·Thomas FolliardLynn J Rothschild
May 16, 2018·Nature Communications·Krishna C SuddalaNils G Walter
Apr 3, 2019·Cold Spring Harbor Perspectives in Biology·Ameya P JalihalNils G Walter
Apr 15, 2021·Proceedings of the National Academy of Sciences of the United States of America·Surajit ChatterjeeNils G Walter
Aug 7, 2021·Nature Communications·Vanessa de JesusBoris Fürtig
Jan 25, 2018·Chemical Reviews·Sujay RayNils G Walter
Jan 1, 2021·Accounts of Chemical Research·Shankar MandalNils G Walter

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Methods Mentioned

BETA
NMR
fluorescence microscopy
FRET
PCR
in vitro transcription
Fluorescence correlation spectroscopy
ISS

Software Mentioned

QuB
GraphPad
Micro
SoftBerry
OriginLab
ImageQuant
KARTS
PyCorrFit
Manager
SiM

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