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Updated: Jun 21, 2026
06:58
Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling
Published on: October 7, 2021
Footprinting analysis of BWYV pseudoknot-ribosome complexes
Marie-Hélène Mazauric1, Jean-Louis Leroy, Koen Visscher
1Laboratoire de Chimie et Biologie Structurales, FRC3115, ICSN-CNRS, Gif-sur-Yvette 91190, France.
RNA (New York, N.Y.)
|July 24, 2009
Summary
To achieve efficient viral -1 ribosomal frameshifting in E. coli, shortening the mRNA spacer near the beet western yellow virus (BWYV) pseudoknot is crucial. This modification maintains pseudoknot integrity with smaller prokaryotic ribosomes.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Viruses utilize programmed -1 ribosomal frameshifting on polycistronic mRNA to regulate gene expression.
- RNA pseudoknots are key elements that induce frameshifting by stalling ribosomes.
- The beet western yellow virus (BWYV) pseudoknot is a well-studied model for eukaryotic frameshifting.
Purpose of the Study:
- To investigate the structural requirements for efficient frameshifting induced by the BWYV pseudoknot in prokaryotic (E. coli) ribosomes.
- To determine optimal mRNA spacer lengths for high frameshifting efficiency in a heterologous system.
- To probe the structural integrity and conformational changes of the BWYV pseudoknot within E. coli ribosomes.
Main Methods:
- Construction and analysis of BWYV pseudoknot variants with modified mRNA spacer lengths.
- Assays to measure frameshifting efficiency in E. coli.
- Chemical probing experiments to assess pseudoknot structure and dynamics in the pretranslocation state.
Main Results:
- Shortening the mRNA spacer by 1 or 2 nucleotides (nt) was necessary to achieve eukaryotic levels of frameshifting efficiency with E. coli ribosomes.
- Modified spacer lengths likely re-establish essential ribosomal contacts or tension with smaller prokaryotic ribosomes.
- Chemical probing revealed a compact pseudoknot structure, with altered nucleotide reactivity indicating conformational changes upon ribosome interaction, including potential melting of stem 1 base pairs in the 1-nt shortened construct.
Conclusions:
- Optimizing the mRNA spacer length is critical for efficient BWYV pseudoknot-mediated frameshifting in prokaryotic systems.
- Spacer length adjustments influence pseudoknot structural integrity and unfolding kinetics during ribosomal translocation.
- These findings provide insights into the adaptation of viral frameshifting elements for expression in different host translation systems.
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