Formation of the first peptide bond: the structure of EF-P bound to the 70S ribosome

Abstract

Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 70S ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNA(i) (fMet-tRNA(i)(fMet)) and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms. EF-P binds to a site located between the binding site for the peptidyl tRNA (P site) and the exiting tRNA (E site). It spans both ribosomal subunits with its amino-terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl-terminal domain positioned next to the anticodon stem-loop of the P site-bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNA(i)(fMet) for the formation of the first peptide bond during translation initiation.

Fig. 1

The structure of EF-P bound to the ribosome. (A) Overview of the E- and P-site tRNAs bound to the 70S ribosome [coordinates were taken from the Protein DataBank (PDB) ID 2j00 and 2j01]. The 50S subunit is colored gray, the 30S subunit is colored yellow, the E-site tRNA is shown in red, and the P-site tRNA is shown in green as a surface representation. Portions of the 70S ribosome were omitted for clarity. (B) Overview of EF-P and P-site tRNA–binding in the 70S ribosome. The 50S, 30S, and P-site tRNA are colored as in (A), and EF-P is shown as a surface representation in shades of magenta to indicate the different domains (I, II, and III) of the protein. (C) Same as (A), but the ribosomal protein L1 is also shown as a surface representation in gold. (D) Same as (B), but L1 is also shown as a surface representation in gold, illustrating the large movement of L1 from its location in (C).

Fig. 2

The interaction interfaces between EF-P, L1, and the initiator tRNA. (A) Ribosomal protein L1 (gold), the P-site tRNA (green), and the mRNA (cyan) are shown as cartoon representations, and EF-P is illustrated as an electrostatic surface, with negatively charged patches displayed in red and positively charged patches within contact of the P-site tRNA displayed in blue. (B) Same as in (A) but rotated 240° with EF-P (magenta) shown as a cartoon representation and the ribosomal protein L1 shown as an electrostatic surface, illustrating the charge complementarity between the interface of EF-P and L1.

Fig. 3

Interactions near the PTC. (A) Overview of the N-terminal domain (NTD) of EF-P and its interactions near the PTC of the large ribosomal subunit. The 23S rRNA is colored gray, with the exception of the P-loop (bluish green) and residues making up the PTC (orange). EF-P is colored magenta, and the acceptor arm of the P-site tRNA is shown in green. EF-P approaches the PTC of the 50S subunit but is too distant to participate directly in catalysis. (B) Unbiased difference Fourier map showing the density for the acceptor arm of the P-site tRNA (green) and the loop of the NTD of EF-P containing the R32 residue. Potential hydrogen bonds between the side chains of EF-P and the tRNA are shown as black dashes.

Fig. 4

Hypusine homology model. (A) Close-up view of the interactions between R32 and K29 of EF-P (magenta) with the CCA end of the tRNA (green) and the 23S rRNA (light blue). Putative hydrogen bonds of R32 with the ribose of C75 and the phosphate of C2064, as well as of K29 with the ribose of G2253, are shown as black dashes. Hydrogen bonds of C74 with G2552 and of C75 with G2251 are also shown. (B) R32 was replaced by a hypothetical model structure of hypusine (light brown). In an elongated conformation, the hypusine side chain could reach into the PTC.

Fig. 5

Interactions between EF-P and the 30S subunit. (A) Overview of the interactions made by the P-site tRNA (green) and C-terminal domain of EF-P (magenta) with the small ribosomal subunit (rRNA, yellow; S7 protein, brown). The two key bases of the small ribosomal subunit that act as a gate between the P and E sites are colored orange. (B) Unbiased difference Fourier map showing the density for the terminal residues of EF-P and C39–C41 of the initiator tRNA. Potential hydrogen bonds between R183 and Y180 and the tRNA are illustrated as black dashes. (C) R138 of EF-P stabilizes the type II A-minor interaction between G1338 and the C41–G29 base pair of the tRNA. Putative hydrogen bonds are shown as black dashes.

Fig. 6

Movement of the L1 stalk. The L1 stalk from the structure of the 70S ribosome with bound EF-P is shown in gold, and the 70S structure from T. thermophilus 70S ribosome with a bound E-site tRNA (PDB ID 2j01) is shown in green. The superposition was based on the entire 23S rRNA between the 70S structures, excluding the L1 stalk. The large ribosomal subunit (gray) and EF-P (magenta) are shown as surface representations. CP indicates the central protuberance of the large ribosomal subunit.