EF-P-Mediated Enhancement of Protein Synthesis: Cyclic Dipeptide Incorporation Using Mono- and Bisaminoacylated tRNAs

Strategies for site-specific incorporation of noncanonical amino acids have advanced substantially, enabling access to proteins containing numerous analogues. That noted, many interesting analogues are not α-L-amino acids, and the incorporation of such compounds often requires the development and use of engineered ribosomes. Recently, elongation factor P (EF-P), a translation factor reported to alleviate ribosomal stalling during the incorporation of contiguous L-proline residues, has been shown to facilitate the incorporation of a diverse range of noncanonical amino acids in the presence of native bacterial ribosomes. However, the specific experimental parameters that enable EF-P-mediated enhancement of extensively modified amino acids remain poorly understood. Presently, we examined EF-P-mediated incorporation of four conformationally constrained cyclic dipeptides into position 24 of human RRM1, a nucleic acid recognition motif, using an in vitro transcription-translation system. We compared the effects of ring substitution and conformational isomerism on protein incorporation. Each was incorporated using mono- and bisaminoacylated tRNACUAPhe in comparison to L-phenylalanine. Supplemental EF-P enhanced the incorporation of all analogues significantly, but varied in magnitude substantially between congeners. Bisaminoacylated tRNAs exhibited distinct profiles relative to their monoaminoacylated counterparts. In the absence of exogenous EF-P, bisaminoacylated tRNAs activated with cyclic dipeptides produced similar or lower protein yields relative to their monoaminoacylated counterparts. In contrast, in the absence of added EF-P bisphenylalanyl-tRNA maintained higher incorporation yields than monophenylalanyl-tRNA, as we have observed previously for many aminoacyl-tRNAs containing α-L-amino acids. In the presence of exogenous EF-P, tRNAs activated with two cyclic dipeptides exhibited disproportionately greater yield enhancement, surpassing the corresponding monoaminoacylated analogues. These findings demonstrate that exogenous EF-P-mediated enhancement is influenced by changes within the local structure of the analogues. Additionally, bisaminoacylated tRNAs have the potential of affording significantly greater protein yields, especially in the presence of supplementary EF-P.