title: mRNA pseudoknot structures can act as ribosomal roadblocks.
abstract: Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknot-like structures. Surprisingly, the structures predicted to be strongest lead only to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction of the frameshifted ribosomes from reaching the downstream stop codon. The stronger the pseudoknot the larger the frameshifting efficiency and the larger its roadblocking effect. The maximal amount of full-length frameshifted product is produced from a structure where those two effects are balanced. Taking ribosomal roadblocking into account is a prerequisite for formulating correct frameshifting hypotheses.
authors: Department of Biology, Ole Maaløes vej 5, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors: Jesper
authors: J
authors: Tholstrup
authors: Lene B
authors: LB
authors: Oddershede
authors: Michael A
authors: MA
authors: Sørensen
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D018965:Frameshifting, Ribosomal; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases