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.

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 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).…”

“…In addition to the ensemble biochemical assays described in Sections 3.4.1 and 3.4.2, we have also developed two smFRET assays that report on aa-tRNA selection and peptidyl transfer Fei et al, 2009;Gonzalez Jr. et al, 2007;Kim et al, 2014), and have adapted these assays for use with ncAA-tRNAs (Figures 3B and C) (Effraim et al, 2009;Gamper et al, 2021). Biophysical smFRET experiments measure the efficiency of FRET (EFRET) between a FRET donor fluorophore and a FRET acceptor fluorophore that have been engineered into specific residue positions in a biomolecule or biomolecular complex of interest.…”

“…The first exception is that 2 μM fMet-(Cy3)tRNA fMet is substituted for 0.5 μM f-[ 35 S]Met-tRNA fMet such that the 70S ribosomes are now the limiting reagent. The second is that either a chemically synthesized, 5'-biotinylated T4gp321-9 mRNA or an in vitro transcribed, T4gp321-20 mRNA that has been pre-hybridized to a 3'-biotinylated DNA oligonucleotide complementary to the first 18 nucleotides of the T4gp321-20 mRNA (Effraim et al, 2009;Gamper et al, 2021) (collectively referred to as Bio-T4gp32 mRNA) is substituted for T4gp321-20 mRNA. 70S ICs from the resulting 70S IC Mix are purified using sucrose density gradient ultracentrifugation as described in Fei (2010).…”

“…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 Schmied et al, 2018;Ward et al, 2019), molecular dynamic (MD) simulations (Englander 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 onenucleotide 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.…”