The central role of tRNA in genetic code expansion

Abstract

Background The development of orthogonal translation systems (OTSs) for genetic code expansion (GCE) has allowed for the incorporation of a diverse array of non-canonical amino acids (ncAA) into proteins. Transfer RNA, the central molecule in the translation of the genetic message into proteins, plays a significant role in the efficiency of ncAA incorporation. Scope of Review Here we review the biochemical basis of OTSs for genetic code expansion. We focus on the role of tRNA and discuss strategies used to engineer tRNA for the improvement of ncAA incorporation into proteins. Major Conclusions The engineering of orthogonal tRNAs for GCE has significantly improved the incorporation of ncAAs. However, there are numerous unintended consequences of orthogonal tRNA engineering that cannot be predicted ab initio. General Significance Genetic code expansion has allowed for the incorporation of a great diversity of ncAAs and novel chemistries into proteins, making significant contributions to our understanding of biological molecules and interactions.

Evolving Mistranslating tRNAs Through a Phenotypically Ambivalent Intermediate inSaccharomyces cerevisiae

“…The efficiency of translation by a tRNA can be reduced at many stages of expression, maturation, and translation. Many of these points of control in the tRNA life cycle are well-described and include reduced transcription by RNA polymerase III, errors in processing, modification, splicing or aminoacylation, decreased affinity for EF-Tu or the ribosome, or an increase in tRNA degradation ( Reynolds et al 2017 ). sup17 ( UGG ) G26A contained a mutation of a dimethylated G to A at position 26.…”

The cellular landscape of mid‐size noncoding RNA

“…However, we propose here that all RNAs between 50–400 nucleotides are a distinct class, the mid‐size noncoding RNAs (Figure ). Many RNA of this type have been the subject of separate reviews that discuss specific functional characteristics (e.g., Decatur, Liang, Piekna‐Przybylska, & Fournier, ; Fang & Guo, ; Lui & Lowe, ; Orioli, ; Reynolds, Vargas‐Rodriguez, Soll, & Crnkovic, ; Tuorto & Lyko, ; Woolford & Baserga, ). In this review, we deviate from earlier work by treating ncRNA that vary from 50–400 as a single class and describe their structural and functional features with a focus on human mncRNAs.…”

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

“…Efficiency of site-specific incorporation of ncAAs via nonsense suppression depends on the catalytic prowess of the OTS [ 3 ], the availability of ncAA (depending on the ncAA intake and its participation in the cellular metabolism, [ 45 ]), the suitability of ncAA-o-tRNA for EF-Tu binding [ 12 ], and the capacity of ncAA-o-tRNA to decode the stop codon of interest [ 26 ] and outcompete the release factor for binding to the same site [ 46 ]. Because each of these processes is usually less efficient than for endogenous translation systems, yields of ncAA-containing proteins are typically low.…”

“…Primary is an engineered aminoacyl-tRNA synthetase (aaRS)•tRNA pair specific for the desired ncAA. This orthogonal translation system (OTS) must operate in a host cell being agnostic of the endogenous aaRSs and tRNAs, while interacting normally with the host translation machinery (e.g., elongation factor and ribosome, [ 12 ]). For these reasons, most OTSs are obtained from organisms of a different domain of life; thus, the OTSs used in Escherichia coli are usually of archaeal or eukaryotic origin [ 3 , 12 ].…”