Drop-off-reinitiation triggered by EF-G-driven mistranslocation and its alleviation by EF-P.

In ribosomal translation, peptidyl transfer occurs between P-site peptidyl-tRNA and A-site aminoacyl-tRNA, followed by translocation of the resulting P-site deacylated-tRNA and A-site peptidyl-tRNA to E and P site, respectively, mediated by EF-G. Here, we report that mistranslocation of P-site peptidyl-tRNA and A-site aminoacyl-tRNA toward E and A site occurs when high concentration of EF-G triggers the migration of two tRNAs prior to completion of peptidyl transfer. Consecutive incorporation of less reactive amino acids, such as Pro and d-Ala, makes peptidyl transfer inefficient and thus induces the mistranslocation event. Consequently, the E-site peptidyl-tRNA drops off from ribosome to give a truncated peptide lacking the C-terminal region. The P-site aminoacyl-tRNA allows for reinitiation of translation upon accommodation of a new aminoacyl-tRNA at A site, leading to synthesis of a truncated peptide lacking the N-terminal region, which we call the 'reinitiated peptide'. We also revealed that such a drop-off-reinitiation event can be alleviated by EF-P that promotes peptidyl transfer of Pro. Moreover, this event takes place both in vitro and in cell, showing that reinitiated peptides during protein synthesis could be accumulated in this pathway in cells.

Genetic code expansion via integration of redundant amino acid assignment by finely tuning tRNA pools.

In all translation systems, the genetic code assigns codons to amino acids as building blocks of polypeptides, defining their chemical, structural and physiological properties. The canonical genetic code, however, utilizes only 20 proteinogenic amino acids redundantly encoded in 61 codons. In order to expand the building block repertoire, this redundancy was reduced by tuning composition of the transfer RNA (tRNA) mixture in vitro. Depletion of particular tRNAs from the total tRNA mixture or its reconstitution with in vitro-transcribed tRNASNNs (S = C or G, N = U, C, A or G) divided a codon box to encode two amino acids, expanding the repertoire to 23. The expanded genetic codes may benefit analysis of cellular regulatory pathways and drug screening.

Consecutive Elongation of D-Amino Acids in Translation.

Recent progress in the field of genetic code reprogramming using a reconstituted cell-free translation system has made it possible to incorporate a wide array of non-proteinogenic amino acids, including N-methyl-amino acids and D-amino acids. Despite the fact that up to ten N-methyl-amino acid residues can be continuously elongated, the successive incorporation of even two D-amino acids into a nascent peptide chain remains a formidable challenge, thus far being nearly impossible. Here we report achievement of continuous D-amino acid elongation by the use of engineered tRNAs and optimized concentrations of translation factors, enabling us to incorporate up to ten consecutive D-Ser residues into a nascent peptide chain. We have also expressed macrocyclic peptides consisting of four or five consecutive D-amino acids consisting of D-Phe, D-Ser, D-Ala, or D-Cys closed by either a disulfide bond or a thioether bond.