Ratchet-like Movements between the Two Ribosomal Subunits: Their Implications in Elongation Factor Recognition and tRNA Translocation

Three-dimensional cryo-electron microscopy (3Dcryo-EM) is uniquely suited to provide information aboutdynamic changes of the ribosome structure in the courseof translation. Usually, a functional ribosome–ligandcomplex is stalled by the addition of an antibiotic that isknown to inhibit a biochemical reaction at a particular intermediate state (see Spahn and Prescott 1996) or substitution of high-energy nucleotide triphosphate compounds, such as GTP, by its nonhydrolyzable analogs. Inmost cases, such compounds bind to the protein factors toprovide a conformation recognizable by the ribosome.Many of the changes are on such a scale that they areeasily detected and measured by fitting of X-ray structures or by comparison of three-dimensional densitymaps showing the ribosome in different states. For example, on the basis of cryo-EM maps with resolutions ranging from 17 Å to 20 Å, EF-G bound to the ribosome in thepresence of fusidic acid (Agrawal et al. 1998, 1999b;Stark et al. 2000) was shown to be in a different conformation than in the X-ray structures, both in its nucleotidefree state (Aevarsson et al. 1994) and in complex withGDP (Czworkowski et al. 1994; al-Karadaghi et al.1996), and the cryo-EM maps allowed a quantitative description of the movements of individual domains(Agrawal et al. 1998, 1999b; Wriggers et al. 2000). An 8Å movement of the top portion of helix 44 of 16S rRNAwithin the smaller 30S subunit was detected by comparing cryo-EM maps of ribosome complexes in the pre- andpost-translocational state (VanLoock et al. 2000). Another most recent example is the observation of a variation in the geometry of the entrance of the tunnel that conducts the polypeptide chain within the 50S subunit withdifferent functional states (Gabashvili et al. 2001)...