The structure of the ribosome with elongation factor G trapped in the posttranslocational state

Abstract

Elongation factor G (EF-G) is a guanosine triphosphatase (GTPase) that plays a crucial role in the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) during translation by the ribosome. We report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with EF-G in the posttranslocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with the tRNA at the peptidyl-tRNA binding site (P site) and with mRNA shed light on the role of these elements in EF-G function. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.

Figure 1

Unbiased difference Fourier electron density maps for (A) EF-G and (B) the ligands fusidic acid (FUS) and GDP with its coordinated Mg²⁺ ion in the ribosome. All figures were made using PyMol ()

Figure 2

EF-G in the post-translocational state of the ribosome. A. Overall view of EF-G in the ribosome. EF-G is shown in reddish-brown, the 50S subunit on top is shown in orange, the 30S subunit below is shown in cyan, P-site tRNA in green, and E-site tRNA in yellow and the mRNA in purple. The decoding center (DC) in the 30S subunit and the peptidyl transferase center (PTC), and the L1 and L10-L12 stalks are indicated as shown.B. Global changes in the 50S subunit as a result of EF-G binding. The mobile regions of the 50S subunit are indicated in teal, and include the L1 stalk (both RNA and protein), the L11 region, and the the L10-L12 stalk. C.The structure of EF-G bound to fusidic acid and GDP in the ribosome. The various domains of EF-G are colored as shown.

Figure 3

Interactions of domain IV of EF-G with tRNA and mRNA. A. Interactions of domain IV of EF-G with P-site tRNA and mRNA. Loop 1 is inserted into the minor groove between P-site tRNA and its codon. In gray are shown elements from a superposition of the 70S structure with A-site tRNA (), showing conformational changes in the mRNA and protein S13 on EF-G binding. Unlike A-site tRNA, domain IV of EF-G does not make extensive interactions with the A-site codon. B. Details of interactions with domain IV in the decoding site. The residues Q500 from loop I and H573 from loop II form a network of interactions with P-site tRNA and each other; Loop 1 has changed conformation relative to its structure in domain IV of the isolated protein (gray) (2BM0 from ref. ), to insert into the minor groove between P-site tRNA and mRNA. Residues S578 and E579 make interactions with the universally conserved A1493 of 16S RNA.

Figure 4

The environment of the nucleotide binding site in EF-G bound to the ribosome in the presence of fusidic acid. A. A superposition shows that the switch I region of EF-G-2 in the GTP-bound form (pink)() has become disordered in the fusidic-acid complex structure. However switch II (red) in the structure is close to the GTP form of EF-G-2, and different from the altered conformation of the GDP form of EF-G in isolation (gray)(2BM0 from ref. ). Similarly, K25, which interacts with the gamma phosphate in the GTP form, remains in that form in this structure rather than adopt the altered conformation (gray) of the GDP form of EF-G. B. Conformational changes of the β–hairpins and 16S movement in domain II relative to the GTP form of EF-G-2 (). This movement is in the same direction as the movement of 16S RNA (cyan) relative to that of the ratcheted ribosome (light blue) that was aligned using the 50S subunit().

Figure 5

Fusidic acid bound to EF-G in the ribosome. A. Interactions of fusidic acid (FA) in a pocket lined by domains of EF-G, showing amino acids whose mutation gives rise to fusidic acid resistance. B. A superposition of switch I from the GTP form of EF-G-2 () shows it would clash with fusidic acid (FA). C. Conformational changes between the fusidic-acid-bound structure compared to the GDP-bound form of EF-G in isolation (2BM0 from ref. ) (gray). F90, switch II of the G domain and domain III are all in a conformation that is different from the GDP form of EF-G.

Figure 6

Interaction of EF-G with the L11 region and the L10-L12 stalk. A. Overview showing that EF-G interacts directly with the L11 RNA (helices 43 and 44) through domain V. The L10-L12 stalk has bent towards the L11 region relative to the structure of the isolated stalk (gray) () as judged by superimposing the globular part of L10. As a result, a copy of the CTD of L12 bridges the G′ domain of EF-G and the NTD of L11 by interacting with both of them. B. Conformational changes in the G′ domain and domain V of EF-G in the GDP-fusidic acid form in the ribosome (reddish-brown) as compared to the GTP form (pink) (). C. Comparison of the structure of EF-G trapped in the post-translocational state of the ribosome with the structure of the 50S subunit bound to micrococcin (). Domain V of EF-G interacts with helices 43 and 44 of 23S RNA that bind L11. A superposition of the micrococcin-bound 50S structure (gray) using all of 23S RNA shows that helices 43 and 44 (gray) superimpose reasonably well on the EF-G ribosome complex, but the NTD of L11 would clash with domain V of EF-G. The CTD of L12 is in the micrococcin-bound 50S structure is also in a very different location and orientation compared to the current structure where it interacts with the G′ domain.