Research Article

Elongation Factor G Bound to the Ribosome in an Intermediate State of Translocation

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Science  28 Jun 2013:
Vol. 340, Issue 6140, 1235490
DOI: 10.1126/science.1235490

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Structured Abstract

Introduction

After peptidyl transfer, the movement of messenger RNA (mRNA) and transfer RNAs (tRNAs) with respect to the ribosome places the next mRNA codon in the A site. This process of translocation proceeds via an intermediate state in which the acceptor ends of the tRNAs have moved with respect to the 50S subunit but not the 30S subunit, to result in A/P and P/E tRNA hybrid states. The guanosine triphosphatase elongation factor G (EF-G) catalyzes the subsequent movement of mRNA and tRNA with respect to the 30S subunit. How EF-G binds to the intermediate state of the ribosome and how this results in guanosine 5′-triphosphate (GTP) hydrolysis and translocation are questions that will be greatly facilitated by a high-resolution structure of the complex.

Embedded Image

Structure of EF-G with GDPCP bound to the ribosome in an intermediate state of translocation. (A) Overview of the structure with a hybrid P/E tRNA. (B) Rotation of the body and swiveling of the head of the 30S subunit (yellow), compared to the canonical state (gray). (C) Catalytic site showing conserved residues around the GDPCP molecule stabilized in an activated conformation.

Methods

Thermus thermophilus ribosomes lacking protein L9 were crystallized in an intermediate state with mRNA, a hybrid P/E tRNA, and EF-G with the nonhydrolyzable GTP analog GDPCP. The structure was solved by molecular replacement and refined to 2.9 Å resolution.

Results

The 50S and 30S ribosomal subunits are rotated relative to each other, as was expected from previous cryo–electron microscopy studies. The L1 stalk on the 50S subunit moves inward to stabilize the P/E hybrid-state tRNA, and atomic details of this interaction can now be seen. Domain IV of EF-G takes up an orientation intermediate between that of the isolated form of EF-G and that of EF-G bound to the ribosome in the fully translocated state. The catalytic center of EF-G shows that key switch regions surrounding the γ-phosphate of GDPCP are clearly visible and therefore ordered. Moreover, key conserved residues in EF-G, including a histidine and an aspartate, change conformation relative to both the isolated and fully translocated structure. These activated conformations appear to be stabilized by the highly conserved sarcin-ricin RNA loop (SRL) of the 50S subunit.

Discussion

Comparison with the posttranslocational state suggests that interactions between the tRNA and L1 stalk are preserved throughout translocation and that these are probably an essential feature of translocation required for stabilization of the hybrid P/E state.

In the isolated structure of EF-G, domain IV, because of its orientation, would largely avoid a clash with A-site tRNA, as would be required for formation of a transient initial complex. In our structure, domain IV partly extends into the A site, which is consistent with the observation that EF-G facilitates translocation at a slow rate even without GTP hydrolysis.

The catalytic center of EF-G has essentially the same structure as that previously observed for elongation factor Tu (EF-Tu), with the highly conserved histidine stabilized by the SRL in an orientation that coordinates a water molecule in position for hydrolysis of GTP. This shows that although EF-Tu and EF-G bind to very different states of the ribosome, the mechanism of activation of GTP hydrolysis is probably the same for these two factors, and possibly for other translational GTPases.

Revealed in Translation

The ribosome, with the help of transfer RNAs (tRNAs), converts the triple genetic code in messenger RNA (mRNA) into protein. Upon decoding of a codon, the mRNA and associated tRNAs must be moved through the ribosome, so that the next codon can be read, with a new charged tRNA taken in at the A (aminoacyl-tRNA) site, the newly extended peptidyl-tRNA moved into the P (peptidyl-tRNA) site, and the deacylated tRNA removed from the exit site in the ribosome (see the Perspective by Rodnina). Crystal structures from Tourigny et al. (p. 1235490), Pulk and Cate (p. 1235970), and Zhou et al. (p. 1236086), variously capture the prokaryotic ribosome during this translocation phase, revealing the hybrid states of the tRNAs and the substantial motions of the 30S ribosomal subunit during the process, the role of elongation factor G, and suggest how the direction and reading frame of the mRNA is maintained.

Abstract

A key step of translation by the ribosome is translocation, which involves the movement of messenger RNA (mRNA) and transfer RNA (tRNA) with respect to the ribosome. This allows a new round of protein chain elongation by placing the next mRNA codon in the A site of the 30S subunit. Translocation proceeds through an intermediate state in which the acceptor ends of the tRNAs have moved with respect to the 50S subunit but not the 30S subunit, to form hybrid states. The guanosine triphosphatase (GTPase) elongation factor G (EF-G) catalyzes the subsequent movement of mRNA and tRNA with respect to the 30S subunit. Here, we present a crystal structure at 3 angstrom resolution of the Thermus thermophilus ribosome with a tRNA in the hybrid P/E state bound to EF-G with a GTP analog. The structure provides insights into structural changes that facilitate translocation and suggests a common GTPase mechanism for EF-G and elongation factor Tu.

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