Insights into Genome Recoding from the Mechanism of a Classic +1-Frameshifting tRNA

Abstract

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

“…The translocation intermediates captured in this work also illustrate how the mRNA frame is preserved to prevent frameshifting events that could produce toxic proteins and premature termination. While the pre-translocation ribosome stabilizes the tRNA-mRNA helix in the decoding center 63, 64, 66 and in the P-site 48, 56, 104 , the thermodynamically labile three-basepair codon-anticodon helix may be destabilized during the transition between these two sites, leading to tRNA slippage and frameshifting 28, 105, 106 . Indeed, recent crystal structure revealed that the tRNA-mRNA base-pairing can be destabilized in the absence of EF-G, if the 30S head is swiveled similarly to that in our Structure V 101 .…”

Mechanistic studies of non-canonical amino acid mutagenesis

“…Note that the protocols reported below for the preparation of the 70S IC Mix, TC Mix, and EF-G Mix and for performing tripeptide synthesis reactions are based on procedures that have been previously published (Blanchard, Kim, et al, 2004; Fei, 2010; Fei et al, 2010; Pavlov & Ehrenberg, 1996). Moreover, these protocols should serve as a general guide; for specific examples of the protocols we have used to prepare the 70S IC Mix, TC Mix, and EF-G Mix and perform tripeptide synthesis reactions in our mechanistic studies of ncAA incorporation, please refer to Effraim et al (2009), Englander et al (2015), Fleisher et al (2018), and the smFRET experiments reported in Gamper et al (2021).…”

“…To clearly show the expected results for a cAA-tRNA that does not exhibit defects in aa-tRNA selection or peptidyl-transfer, the data shown for the L1-L9 smFRET assay correspond to a proline (Pro) that has been acylated onto a wildtype tRNA Pro rather than onto a +1FS-inducing tRNA. The data and corresponding data figure panels are from Gamper et al (2021).…”

“…Specifically, determining when, where, and how during translation the TM fails to incorporate particular ncAAs can inform rational design-or directed evolution approaches aimed at overcoming such mechanistic obstacles and expanding the range of ncAAs that the TM can use. Motivated by this principle, we and others are using biochemistry (Aleksashin et al, 2019; Effraim et al, 2009; Englander et al, 2015; Fleisher et al, 2018; Gamper et al, 2021; Heckler et al, 1988), rapid kinetics (Gamper et al, 2021; Liljeruhm et al, 2019), single-molecule biophysics (Effraim et al, 2009; Gamper et al, 2021), structural biology (Melnikov et al, 2019; Schmied et al, 2018; Ward et al, 2019), molecular dynamic (MD) simulations (Englander et al, 2015; Maini, Chowdhury, et al, 2015), computational analyses (Walker et al, 2020), and other mechanistic tools to explore the limits of ncAA incorporation by the TM.…”

“…The findings suggested that engineering specific features of the PTC and/or incoming aa-tRNA can facilitate incorporation of D-aas and, very likely, other ncAAs that might arrest translation through a similar mechanism. In a second example, we have investigated the mechanism through which a tRNA containing a one-nucleotide insertion mutation in its anticodon loop induces the bacterial TM to undergo a highly efficient +1 frameshifting (FS) event at a specific quadruplet-nucleotide codon, thereby enabling the robust incorporation of an ncAA by the TM (Gamper et al, 2021). In this study, we found that the tRNA induces +1FS at the quadruplet codon by manipulating a specific conformational rearrangement of the ribosomal small, or 30S, subunit that is necessary for translocation of the ribosome along the mRNA.…”