Precise tuning of bacterial translation initiation by non-equilibrium 5'-UTR unfolding observed in single mRNAs.

Nucleic Acids Res

Single-Molecule Analysis Group, Department of Chemistry and Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI 48109, USA.

Published: August 2022

Noncoding, structured 5'-untranslated regions (5'-UTRs) of bacterial messenger RNAs (mRNAs) can control translation efficiency by forming structures that either recruit or repel the ribosome. Here we exploit a 5'-UTR embedded preQ1-sensing, pseudoknotted translational riboswitch to probe how binding of a small ligand controls recruitment of the bacterial ribosome to the partially overlapping Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5'-UTR during mRNA accommodation into the mRNA binding cleft. Ligand binding to the riboswitch stabilizes the structure to both antagonize 30S recruitment and accelerate 30S dissociation. Proximity of the 5'-UTR and stability of the SD:anti-SD interaction both play important roles in modulating the initial 30S-mRNA interaction. Finally, depletion of small ribosomal subunit protein S1, known to help resolve structured 5'-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5'-UTR during accommodation provides a mechanistic understanding of how translation efficiency is governed by riboswitches and other dynamic structure motifs embedded upstream of the translation initiation site of bacterial mRNAs.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9410914PMC
http://dx.doi.org/10.1093/nar/gkac635DOI Listing

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