Footprinting analysis of BWYV pseudoknot-ribosome complexes

Marie Hélène Mazauric, Jean Louis Leroy, Koen Visscher, Satoko Yoshizawa, Dominique Fourmy

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. When the ribosome encounters the pseudoknot barrier that resists unraveling, transient mRNA-tRNA dissociation at the decoding site, results in a shift of the reading frame. The eukaryotic frameshifting pseudoknot from the beet western yellow virus (BWYV) has been well characterized, both structurally and functionally. Here, we show that in order to obtain eukaryotic levels of frameshifting efficiencies using prokaryotic Escherichia coli ribosomes, which depend upon the structural integrity of the BWYV pseudoknot, it is necessary to shorten the mRNA spacer between the slippery sequence and the pseudoknot by 1 or 2 nucleotides (nt). Shortening of the spacer is likely to re-establish tension and/or ribosomal contacts that were otherwise lost with the smaller E. coli ribosomes. Chemical probing experiments for frameshifting and nonframeshifting BWYV constructs were performed to investigate the structural integrity of the pseudoknot confined locally at the mRNA entry site. These data, obtained in the pretranslocation state, show a compact overall pseudoknot structure, with changes in the conformation of nucleotides (i.e., increase in reactivity to chemical probes) that are first "hit" by the ribosomal helicase center. Interestingly, with the 1-nt shortened spacer, this increase of reactivity extends to a downstream nucleotide in the first base pair (bp) of stem 1, consistent with melting of this base pair. Thus, the 3 bp that will unfold upon translocation are different in both constructs with likely consequences on unfolding kinetics.

Original languageEnglish (US)
Pages (from-to)1775-1786
Number of pages12
JournalRNA
Volume15
Issue number9
DOIs
StatePublished - Sep 1 2009

    Fingerprint

Keywords

  • BWYV
  • Frameshifting
  • Translation
  • Translocation
  • mRNA

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Mazauric, M. H., Leroy, J. L., Visscher, K., Yoshizawa, S., & Fourmy, D. (2009). Footprinting analysis of BWYV pseudoknot-ribosome complexes. RNA, 15(9), 1775-1786. https://doi.org/10.1261/rna.1385409