Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

Abstract

Ribosome-dependent protein biosynthesis is an essential cellular process mediated by transfer RNAs (tRNAs). Generally, ribosomally synthesized proteins are limited to the 22 proteinogenic amino acids (pAAs: 20 L-α-amino acids present in the standard genetic code, selenocysteine, and pyrrolysine). However, engineering tRNAs for the ribosomal incorporation of non-proteinogenic monomers (npMs) as building blocks has led to the creation of unique polypeptides with broad applications in cellular biology, material science, spectroscopy, and pharmaceuticals. Ribosomal polymerization of these engineered polypeptides presents a variety of challenges for biochemists, as translation efficiency and fidelity is often insufficient when employing npMs. In this Review, we will focus on the methodologies for engineering tRNAs to overcome these issues and explore recent advances both in vitro and in vivo. These efforts include increasing orthogonality, recruiting essential translation factors, and creation of expanded genetic codes. After our review on the biochemical optimizations of tRNAs, we provide examples of their use in genetic code manipulation, with a focus on the in vitro discovery of bioactive macrocyclic peptides containing npMs. Finally, an analysis of the current state of tRNA engineering is presented, along with existing challenges and future perspectives for the field.

Tuning tRNAs for improved translation

“…The flexibility of the anticodon stem-loop, largely influenced by base pair 31:39 and non-canonical interactions between nucleotide 32:38 ( Auffinger and Westhof, 1999 ), impacts this decoding efficiency and codon recognition specificity ( Olejniczak et al, 2005 ; Ledoux and Uhlenbeck, 2008 ; Sigal et al, 2024 ). “Stiffer” anticodon arms result from G:C base pairs at these positions, generally distorting the canonical anticodon loop conformation and disrupting the binding of GC-rich codons ( Olejniczak et al, 2005 ; Olejniczak and Uhlenbeck, 2006 ; Murakami et al, 2009 ), whereas weaker interactions, including the rare A32:U38 interaction, allows easier codon-anticodon base pairing ( Ledoux et al, 2009 ; Schmeing et al, 2011 ; Grosjean and Westhof, 2016 ).…”

“…However, the exact impact of adding or removing a modification across different tRNAs is not well understood, and some unmodified tRNAs can still function ( Phizicky and Alfonzo, 2010 ; Lorenz et al, 2017 ). One heuristic approximation is that GC-rich decoding tRNAs are modified to reduce binding affinity whereas AU-rich decoding tRNAs are modified to improve codon binding ( Uhlenbeck and Schrader, 2018 ; Sigal et al, 2024 ). One recent example improved the UAG decoding efficiency of a previously engineered opt tRNA CUA Tyr ( Young et al, 2010 ) by replacing adenine with 2, 6-diaminopurine (D) at positions 31 and 36 ( Mala and Saraogi, 2022 ).…”

“…However, these strategies are not compatible with screening vast libraries for structurally unique or medically relevant peptides, which is a possibility in a ribosome-based ( Yamagishi et al, 2011 ; Goto and Suga, 2021 ; Katoh and Suga, 2022a ). Through tRNA engineering, hundreds of traditionally ribosome-incompatible substrates have been permitted for ribosomal installation with varying levels of success ( Katoh and Suga, 2022a ; Sigal et al, 2024 ). Additionally, nearly 10% and 50% of pathogenic genetic conditions result from aberrant stop codons ( Mort et al, 2008 ) and missense mutations ( Katsonis et al, 2014 ; Stenson et al, 2017 ), respectively.…”

“…Briefly, the former objective optimizes aaRS:tRNA interactions to ensure orthogonality and production of the desired aa-tRNAs ( Soye et al, 2015 ; Tamaki et al, 2018 ; Melnikov and Söll, 2019 ; Krahn et al, 2020b ; Ganesh and Maerkl, 2022 ; Kim et al, 2024 ). The latter objective optimizes aa-tRNA interactions with elongation factors, mRNA, and the ribosome to mediate initiation ( Tharp et al, 2021b ; Katoh and Suga, 2023a ; Katoh and Suga, 2023c ) or elongation fidelity ( Uhlenbeck and Schrader, 2018 ; Sigal et al, 2024 ). Our review addresses tRNA engineering principles for the latter processes as a roadmap for modulating tRNA incorporation into the ribosome.…”

“…with varying levels of success (Katoh and Suga, 2022a;Sigal et al, 2024). Additionally, nearly 10% and 50% of pathogenic genetic conditions result from aberrant stop codons (Mort et al, 2008) and missense mutations (Katsonis et al, 2014;Stenson et al, 2017), respectively.…”