Abstract
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 tRNAPro affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNAPro is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNAPro isoacceptors and tRNAfMet in Escherichia coli, and the D-arm of tRNAfMet 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.
Highlights
The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines
To identify the elements of peptidyl-Pro-tRNAPro steering the interactions with EF-P in the ribosome complex, we used a reconstituted in-vitro translation system coupled with flexizyme technology, referred to as the flexible in-vitro translation (FIT) system[23]
We conclude that the function of EF-P critically depends on two constraints: (1) the P and A sites of the peptidyl transferase centre must be occupied by a peptidyl-Pro-tRNA and a sufficiently unreactive substrate carrying Pro or other secondary amino acid, respectively; and (2) the P-site tRNA must bear the tRNAPro D-loop closed by a stable stem regardless of the sequence of the remaining part of the tRNA molecule; among E. coli tRNAs, only tRNAPro and tRNAfMet share such sequences
Summary
The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The structure of Thermus thermophilus 70S ribosomes in complex with EF-P and tRNAfMet revealed that EF-P binds between the exit (E) and the peptidyl (P) sites of the ribosome and interacts with the phosphate backbone of tRNAfMet at the acceptor stem, D-arm and the anticodon stem (Fig. 1a,b) These interactions may stabilize tRNAfMet in the P site[20], the mechanism for the acceleration of peptide bond formation between fMet–tRNAfMet and Pmn remained unclear. The structure of the ribosome in complex with EF-P and its cellular substrate Pro-tRNAPro is currently not available, and the potential contribution of interactions between EF-P and the mRNA codon in the E site is uncertain[20,21] It is not known whether interactions between EF-P and peptidylPro-tRNAPro accelerate Pro-Pro formation. The action of EF-P is promoted by the presence of a Pro and the recognition elements in the P-site tRNA, which explains the specificity of the factor for polyproline motifs
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