title: Footprinting analysis of BWYV pseudoknot-ribosome complexes.
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.
authors.affiliation: Laboratoire de Chimie et Biologie Structurales, FRC3115, ICSN-CNRS, Gif-sur-Yvette 91190, France.
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Mazauric
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Leroy
authors.forename: Koen
authors.initials: K
authors.lastname: Visscher
authors.forename: Satoko
authors.initials: S
authors.lastname: Yoshizawa
authors.forename: Dominique
authors.initials: D
authors.lastname: Fourmy
mesh_terms: D017122:Bacteriophage T4 / Q000235:genetics; D001483:Base Sequence; D029968:Escherichia coli Proteins / Q000378:metabolism; D018965:Frameshifting, Ribosomal / Q000502:physiology; D017793:Luteovirus / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D023302:Protein Footprinting / Q000379:methods; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D012367:RNA, Viral