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Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
Published on: May 10, 2018
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Specific length and structure rather than high thermodynamic stability enable regulatory mRNA stem-loops to pause
Chen Bao1, Mingyi Zhu1, Inna Nykonchuk1
1Department of Biochemistry & Biophysics at School of Medicine and Dentistry and Center for RNA Biology, University of Rochester, Rochester, NY, USA.
Nature Communications
|February 22, 2022
Summary
Specific mRNA structures, not just stability, stall ribosomes and cause frameshifting. This finding impacts understanding translation regulation and identifying novel regulatory mRNA elements.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Translating ribosomes unwind mRNA secondary structures during elongation.
- Certain mRNA stem-loops can inhibit translation elongation and induce programmed ribosomal frameshifting.
Purpose of the Study:
- To investigate if the thermodynamic stability of three basepairs, unwound by ribosomes, is critical for inducing ribosome pauses.
- To understand the mechanisms by which mRNA stem-loops stall translation.
Main Methods:
- Mutagenesis experiments
- Biochemical assays
- Single-molecule experiments
Main Results:
- Frameshift-inducing mRNA stem-loops (E. coli dnaX, HIV gag-pol) hinder A-site tRNA binding and slow ribosome translocation 15-20 fold.
- Unwinding of the first three basepairs near the mRNA entry channel causes only a 2-3 fold slowdown.
- Specific mRNA stem-loop length and structure, rather than high thermodynamic stability, cause translation stalling via inhibitory ribosome interactions.
Conclusions:
- Ribosome stalling and frameshifting are regulated by specific mRNA stem-loop features, not solely thermodynamic stability.
- These findings provide a basis for transcriptome-wide studies of translation and discovery of novel regulatory mRNA elements.
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