Programmed –1 Frameshifting by Kinetic Partitioning during Impeded Translocation

Abstract

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.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation

“…Previous work has shown that −1 frameshifting occurs during translocation, and is the result of uncoupling of translocation and back-rotation of the small subunit, resulting in an unusual rotated state (Caliskan et al, 2014; Chen et al, 2014). Thus, our data strongly suggest that the Fap7-regulated release of Dim1 is a functional test for correct adoption of the rotated state by nascent 40S subunits and further emphasize the importance of quality control during ribosome assembly.…”

“…Careful kinetic and thermodynamic measurements have demonstrated a near-equilibrium for the different ribosome conformations (Munro et al, 2009). Interestingly, programmed frameshifting occurs by manipulation of the translocation process (Caliskan et al, 2014; Chen et al, 2014). Both of these observations underscore the importance and delicacy of this process.…”

Clarifying the Translational Pausing Landscape in Bacteria by Ribosome Profiling

“…SD motifs have well-characterized roles in frameshifting in bacteria: in the dnaX gene, an internal SD motif contributes to −1 frameshifting at a slippery sequence followed by an mRNA hairpin (Larsen et al, 1994). In vitro studies on this system have shown that the downstream hairpin blocks translocation (Caliskan et al, 2014; Chen et al, 2014), resulting in a kinetic pause; this in turn allows different codons and reading frames to be sampled on the slippery sequence (Caliskan et al, 2015; Yan et al, 2015). In the metastable state where the ribosome slips on the message, the SD motif stabilizes the interaction with the mRNA in a new position.…”

Dual DNA rulers reveal an ‘mRNA looping’ intermediate state during ribosome translocation

“…Among indirect translocation assays, the tandem LC/MS/MS analysis of oligopeptide composition deduces the ribosome reading frame based on the synthesized peptide [8]. Fluorescently labeled tRNA binding/moving events reveal the translating codons [9], and the puromycin reactivity assay is the conventional method for confirming the A-site vacancy resulting from translocation [10]. None of these methods can reveal the movement at the 5ʹ end of the mRNA that is entering the ribosome.…”