Evolutionary tuning impacts the design of bacterial tRNAs for the incorporation of unnatural amino acids by ribosomes

Abstract

In order to function on the ribosome with uniform rate and adequate accuracy, each bacterial tRNA has evolved to have a characteristic sequence and set of modifications that compensate for the differing physical properties of its esterified amino acid and its codon-anticodon interaction. The sequence of the T-stem of each tRNA compensates for the differential effect of the esterified amino acid on the binding and release of EF-Tu during decoding. The sequence and modifications in the anticodon loop and core of tRNA impact the codon-anticodon strength and the ability of the tRNA to bend during codon recognition. These discoveries impact the design of tRNAs for the efficient and accurate incorporation of unnatural amino acids into proteins using bacterial translation systems.

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

“…Variations in the thermodynamic contribution of these two interactions compensate for each other so that the affinity of EF-Tu•GTP for all proteogenic aminoacyl-tRNAs is similar, a feature thought to be of paramount importance for effective and accurate decoding of messenger RNAs by the ribosome ( 53 ). The variations in the affinity of EF-Tu•GTP for the tRNA body of aminoacyl-tRNAs are nearly exclusively determined by the first three base pairs of the T stem ( 54 ). The role of these six nucleotide residues in determining the relative binding affinity of EF-Tu•GTP for the tRNA body of aminoacyl-tRNAs is universally conserved in bacteria in spite of extensive sequence diversity in this portion of the T stem ( 55 ).…”

“…The thermodynamic contribution of each of the three base pairs is additive enabling similar affinities to be reached with various nucleotide combinations ( 52 ). The contribution of the esterified amino acid to the binding affinity of EF-Tu•GTP for aminoacyl-tRNAs is one of the lowest for glycine, and consequently, this is compensated for by one of the highest affinities for the tRNA Gly bodies ( 52 , 54 ). In S. aureus , the proteogenic P1 and P2 isoacceptors display, as expected, high-affinity base-pair combinations at the first three positions of the T stem (G 49 •U 65 , C 50 -G 64 or U 50 -A 64 and G 51 -C 63 , respectively, Figure 1C ) ( 15 , 55 ).…”

Versatility of Synthetic tRNAs in Genetic Code Expansion

“…Attempts to advance ncAA delivery by tRNA Pyl engineering include those aiming to improve its compatibility with the cellular machinery of the host. In E. coli , tRNA Pyl was evolved by targeting the EF-Tu-binding regions [ 87 ], although the optimizing mutations present in tRNA Pyl OPT may be more suitable for the delivery of one particular ncAA and less for the other (e.g., N ε -acetyllyine vs. 3-cyano-phenylalanine) [ 88 ]. The need to separately evolve an o-tRNA for a variety of “cognate” ncAAs or a variety of anticodons may require tunable binding by EF-Tu and the ribosome; while the stability of the EF-Tu•ncAA-tRNA complex reflects additive contributions by the ncAA and T-stem base pairs of the o-tRNA [ 80 , 89 ], the strength of codon–anticodon binding correlates with the nucleotide composition of the tRNA core [ 90 ].…”

Packing the Standard Genetic Code in its box: 3-dimensional late Crick wobble

“…“Late” implies that wobble was deferred, being preceded by unique triplet pairing assignments. Unique base pairing does not require support from a highly evolved allosteric ribosome (Moazed and Noller 1990; Ogle et al 2001), or a specific, highly optimized tRNA anticodon loop-and-stem structure (Yarus 1982; Uhlenbeck and Schrader 2018), or control of isomerization in wobble-paired bases (Westhof et al 2019). Accurate Crick wobble (Crick 1966) would therefore likely be a later, more modern code refinement.…”