Structured m RNA induces the ribosome into a hyper‐rotated state

Abstract

During protein synthesis, mRNA and tRNA are moved through the ribosome by the process of translocation. The small diameter of the mRNA entrance tunnel only permits unstructured mRNA to pass through. However, there are structured elements within mRNA that present a barrier for translocation that must be unwound. The ribosome has been shown to unwind RNA in the absence of additional factors, but the mechanism remains unclear. Here, we show using single molecule F€ orster resonance energy transfer and small angle X-ray scattering experiments a new global conformational state of the ribosome. In the presence of the frameshift inducing dnaX hairpin, the ribosomal subunits are driven into a hyper-rotated state and the L1 stalk is predominantly in an open conformation. This previously unobserved conformational state provides structural insight into the helicase activity of the ribosome and may have important implications for understanding the mechanism of reading frame maintenance.

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for −1 ribosomal frameshifting stimulation

“…( ii ) The frameshifting efficiencies for SRV-1 pseudoknots were previously measured for an eukaryotic ribosome (using a commercially available in vitro translation system)19. ( iii ) During ribosomal frameshifting, a ribosome in contact with a downstream frameshifting stimulatory mRNA structure can pause seconds to minutes and change its conformation3031323334 to facilitate the unwinding of highly stable mRNA structures including pseudoknots. The distances between the folded state and unfolding transition state ( X ‡ ) showed no correlation with frameshifting efficiencies and are about 2 nm for all pseudoknots (Fig.…”

“…Significant advances have been recently made in understanding the conformational dynamics and kinetic partitioning of the prokaryotic ribosome complex, and (un)binding dynamics of tRNA and elongation factor G (EF-G) during the −1 frameshifting process3031323334. A mechanistic picture emerging from the conformational and compositional dynamics studies is that a downstream mRNA pseudoknot or hairpin structure may trap the ribosome at a rotated pre-translocation conformation and slow down the subsequent translation steps in the 0 frame.…”

Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions

“…This non-canonical conformation may be hyper-rotated (Qin et al, 2014) or represent a translocation intermediate (Tourigny et al, 2013). The rotated state, with its weakened ribosome-tRNA-mRNA interactions, is key to allowing the mRNA rearrangements that promote bypassing.…”

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

“…For the mRNA to become mobile, the codon-anticodon duplex must be disrupted. This may be brought about by a conformational rearrangement of the ribosome, for example, the formation of a highly rotated chimeric intermediate similar to that formed during EF-G-catalysed translocation [24][25][26] or frameshifting 27 . Although the codonanticodon interactions are weakened, the retention of peptidyltRNA Gly in the ribosome is ensured by the interactions of the nascent peptide with the ribosome, presumably through both unspecific anchoring of the N-terminal part of gp60 emerging from the ribosome peptide exit tunnel and specific interactions of the part of nascent peptide residing in the exit tunnel.…”

“…The 5 0 SL alone or in combination with the SL element in the 5 0 gap may interact with protein L9, thereby contributing to the processivity NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5459 ARTICLE of sliding or the fidelity of landing codon selection. Finally, it may affect the function of L1, the protein which influences the retention of tRNA 29 and the formation of a particular, overrotated conformation of the ribosome 27 . Previous in-vivo experiments did not implicate the region immediately upstream the gap in the mechanism of bypassing either because the effect of specific mutations was not tested directly or because in vivo the effect is mimicked by contributions of other elements, which are more important under in-vivo conditions.…”