The energy landscape of −1 ribosomal frameshifting

Abstract

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3′ messenger RNA (mRNA) structures, but how these mRNA structures enhance −1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal −1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

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

“…2D, F) were 5-and 7-fold shorter, respectively, than those measured in ribosomes programmed with dnaX_Slip mRNA. In agreement with previously published results (Caliskan et al, 2017;Chen et al, 2014;Choi et al, 2020;Kim et al, 2014), our data demonstrate that dnaX FSS positioned near the mRNA channel entrance strongly inhibits mRNA/tRNA translocation ( Fig. 2E-F).…”

“…(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).…”

“…We asked whether these bacterial and viral mRNA sequences, which form stem-loops of the similar lengths, act by a similar mechanism. In agreement with previous studies (Caliskan et al, 2014;Caliskan et al, 2017;Chen et al, 2014;Choi et al, 2020;Kim et al, 2014), we detected FSS-induced inhibition of tRNA/mRNA translocation. We also observed that FSSs inhibit A-site tRNA binding.…”

“…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).…”

A cytosine-to-uracil change within the programmed -1 ribosomal frameshift signal of SARS-CoV-2 results in structural similarities with the MERS-CoV signal

“…As the C-to-U transition is not a frequent change introduced by the polymerase, it is possible that it originates from RNA editing by a cytosine deaminase 35,42,43 . Biophysical single-molecule assays and kinetics studies on ensembles revealed how the interplay between the conformational plasticity of RNA-inducing frameshifting structures and the dynamics of the ribosome play a critical role in the -1 PRF mechanism [50][51][52][53][54] . Interestingly, the type 1 change results in the substitution of a G-C Watson-Crick pair in the functional core of the SARS-CoV-2 pseudoknot with a wobble G-U pair.…”