EF-G catalyzed translocation dynamics in the presence of ribosomal frameshifting stimulatory signals

Abstract

Programmed -1 ribosomal frameshifting (-1PRF) is tightly regulated by messenger RNA (mRNA) sequences and structures in expressing two or more proteins with precise ratios from a single mRNA. Using single-molecule fluorescence resonance energy transfer (smFRET) between (Cy5)EF-G and (Cy3)tRNALys, we studied the translational elongation dynamics of -1PRF in the Escherichia coli dnaX gene, which contains three frameshifting signals: a slippery sequence (A AAA AAG), a Shine-Dalgarno (SD) sequence and a downstream hairpin. The frameshift promoting signals mostly impair the EF-G-catalyzed translocation step of the two tRNALys and the slippery codons from the A- and P- sites. The hairpin acts as a road block slowing the translocation rate. The upstream SD sequence together with the hairpin promotes dissociation of futile EF-G and thus causes multiple EF-G driven translocation attempts. A slippery sequence also helps dissociation of the EF-G by providing alternative base-pairing options. These results indicate that frameshifting takes place during the repetitive ribosomal conformational changes associated with EF-G dissociation upon unsuccessful translocation attempts of the second slippage codon from the A- to the P- sites.

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

“…To that end, ribosomes bound with dnaX_Slip mRNA and P-site N-Ac-Val-tRNA Val were incubated with EF-G•GTP, EF-Tu•GTP, Lys-tRNA Lys , Arg-tRNA Arg (binds in 0 frame) and [ 3 H]Glu-tRNA Glu (binds in -1 frame). Consistent with previous publications (Caliskan et al, 2017;Kim and Tinoco, 2017;Larsen et al, 1997), we observed frameshifting efficiency of ~60% (Suppl. Fig.…”

“…(i) slippage of the single P-site tRNA when the A site remains vacant, or (ii) frameshifting of both P-site and A-site tRNAs when two tRNAs are bound to the ribosome (Caliskan et al, 2017;Korniy et al, 2019). Our finding that the dnaX FSS slows the rate of ribosome translocation by ~10-fold is also in excellent agreement with a number of previous reports (Caliskan et al, 2017;Chen et al, 2014;Choi et al, 2020;Kim et al, 2014;Kim and Tinoco, 2017).…”

“…The γ subunit is produced by a -1 PRF event that occurs with 50-80% efficiency. We chose dnaX mRNA because it is one of the most extensively studied -1 PRF systems that has been investigated using both ensemble and single-molecule kinetic approaches (Caliskan et al, 2017;Chen et al, 2014;Choi et al, 2020;Kim et al, 2014;Kim and Tinoco, 2017).…”

“…1). Mutations in the slippery sequence of dnaX were shown to decrease frameshifting efficiency to low ( 5%) or undetectable levels (Bock et al, 2019;Caliskan et al, 2017;Kim and Tinoco, 2017;Larsen et al, 1997;Tsuchihashi and Brown, 1992). The FSS from HIV also stalls the ribosome in the NR conformation.…”

Translational accuracy of a tethered ribosome

“…Indeed, for +1 frameshifting, this interaction causes the release of the deacylated E-site tRNA, destabilizing the ribosome complex and affecting P-site tRNA slippage 20,21 . Both types of frameshifting are known to involve base pairing between the SD sequence and the ASD sequence on the 16S rRNA to increase frameshifting efficiency, but the mechanisms of these two types of frameshifting event are different 20,[22][23][24][25] .…”