Abstract
In the past two decades, tremendous efforts for increasing the efficiency of ribosomal incorporation of backbone-modifying nonproteinogenic amino acids (npAAs) have been made and given significant successes. For instance, the use of an engineered body sequence of transfer RNA (tRNA), known as tRNAPro1E2, that efficiently recruits EF-Tu and EF-P significantly improves consecutive incorporation of npAAs, giving a notion that certain protein factors paired with right tRNAs can enhance their incorporation efficiency. However, the consecutive incorporation of certain npAAs, e.g.N-methyl-l-leucine, remains more challenging. Here we have explored Escherichia coli ATP-binding cassette family-F proteins (EttA, Uup, YbiT, and YhsS) and RbbA for a possibility of enhancing the translation efficiency for such npAAs since these proteins are known to alleviate nascent peptide-dependent translation arrest. Indeed, among them the presence of Uup increases the translation level of model peptides bearing two consecutive npAAs by an average of 1.7-fold for 12 kinds of npAAs and that of a macrocyclic peptide bearing d-α-amino, N-methyl-l-α-amino, and β-amino acids by 1.8-fold. Moreover, the combination of EF-P and Uup further enhances the incorporation of npAAs charged on tRNAPro1E2, demonstrating a four-fold enhancement for two consecutive incorporations of N-methyl-l-leucine.