Essential structural elements in tRNA Pro for EF-P-mediated alleviation of translation stalling

The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNA Pro affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNA Pro is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNA Pro isoacceptors and tRNA fMet in Escherichia coli , and the D-arm of tRNA fMet is essential for EF-P-induced acceleration of fMet–puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm.