Crystal Structures of EF-G–Ribosome Complexes Trapped in Intermediate States of Translocation

Introduction: One of the most critical and complex steps of protein synthesis is the coupled translocation of mRNA and tRNAs (mRNA and tRNAs) through the ribosome, catalyzed by the guanosine triphosphatase (GTPase) elongation factor EF-G. Although several of the main steps have been identified, the underlying molecular mechanisms of translocation are poorly understood. A central question is how structural rearrangements in the ribosome are coupled to movement of mRNA and tRNA. Methods: We trapped and crystallized complexes of Thermus thermophilus ribosomes bound with EF-G, mRNA, and tRNA, using the antibiotic fusidic acid (which prevents release of EF-G after GTP hydrolysis) or the nonhydrolyzable GTP analog GDPNP, in intermediate states of translocation. Their crystal structures were determined to resolutions from 3.5 to 4.1 Å. Results: The structures of the fusidic acid complex (Fus) and two GDPNP complexes (GDPNP-I and GDPNP-II) reveal conformational changes occurring during intermediate states of translocation, including large-scale (15° to 18°) rotation of the 30S subunit head and 3° to 5° rotation of the 30S body. In all complexes, the tRNA acceptor end has moved from the 50S subunit P site to the 50S E site, while the anticodon stem loop (ASL) and mRNA move with the head of the 30S subunit to positions between the P and E sites, forming chimeric pe*/E intermediate states. The elongated, mobile domain IV of EF-G moves to contact the head of the 30S subunit and the backbone of the mRNA. Two universally conserved bases of 16S rRNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. In the GDPNP complexes, structuring of the conserved switch loop I segment, which was disordered in previous structures, completes the cage that encloses GDPNP and fixes the relative geometry of EF-G domains I, III, and V. In the Fus complex, the position of fusidic acid overlaps that of switch loop I, stabilizing contacts between domains I and III that are normally made by the structured switch loop. Conclusion: Our structures capture intermediate states of the rate-limiting step of translocation, in which movement of the tRNA ASL and mRNA is coupled to rotational movement of the 30S subunit head. Slippage of the translational reading frame during reverse rotation of the head during translocation may be prevented by intercalation of bases C1397 and A1503 of 16S rRNA, which project from the body of the 30S subunit, between mRNA bases. The antibiotic fusidic acid appears to stabilize binding of EF-G to the ribosome in the GDP state by mimicking the structure of the conserved core of switch loop I of EF-G in the GTP state. 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. Crystal structures reveal how messenger RNA and transfer RNAs transition through the prokaryotic ribosome during translation. [Also see Perspective by Rodnina] Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA–EF-G complexes trapped with β,γ-imidoguanosine 5′-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.