Programmed –1 Frameshifting by Kinetic Partitioning during Impeded Translocation

Programmed –1 ribosomal frameshifting (−1PRF) is an mRNA recoding event utilized by cells to enhance the information content of the genome and to regulate gene expression. The mechanism of –1PRF and its timing during translation elongation are unclear. Here, we identified the steps that govern –1PRF by following the stepwise movement of the ribosome through the frameshifting site of a model mRNA derived from the IBV 1a/1b gene in a reconstituted in vitro translation system from Escherichia coli. Frameshifting occurs at a late stage of translocation when the two tRNAs are bound to adjacent slippery sequence codons of the mRNA. The downstream pseudoknot in the mRNA impairs the closing movement of the 30S subunit head, the dissociation of EF-G, and the release of tRNA from the ribosome. The slippage of the ribosome into the –1 frame accelerates the completion of translocation, thereby further favoring translation in the new reading frame. [Display omitted] •Kinetics of –1 ribosomal frameshifting are monitored at single-codon resolution•Frameshifting occurs when the backward movement of the 30S subunit head is impeded•Two tRNAs at the XXXYYYZ mRNA sequence are stalled in chimeric POST states•The shift to the –1 reading frame occurs prior to EF-G release from the ribosome Programmed ribosomal frameshifting is an mRNA recoding process that allows cells to maximize gene expression. Kinetic deconstruction of this process reveals that the binding of tRNAs to adjacent slippery sequence codons of the mRNA and the resulting pseudoknot impair movement of the 30S ribosome and slip it to the −1 position into a new translational frame.