The two-subunit organization of the ribosome was first linked to its structural dynamics by Spirin [6
] and Bretscher [5
] who independently predicted that translocation, the coupled movement of mRNA and tRNA throught the ribosome, is based on intersubunit movement. In the late 1980s, chemical footprinting experiments showed that translocation takes place in two consecutive steps [7
]. In the first step, the tRNAs move on the 50S subunit, leading to formation of a hybrid-state intermediate (). In the second step, EF-G·GTP catalyzes their movement, coupled to that of mRNA, on the 30S subunit. This result, together with sedimentation and neutron scattering studies ([8
] and references therein), was suggestive of a role for intersubunit movement in translocation.
The first direct evidence for independent movement of the ribosomal subunits during translocation came from the cryo-EM studies of Frank and Agrawal [10
]. Ribosome complexes containing elongation factor EF-G bound with either a non-hydrolysable analog of GTP (GDPNP) or GDP and fusidic acid (an antibiotic that prevents release of EF-G following GTP hydrolysis) were found to have an altered conformation, in which the small subunit was rotated counter-clockwise with respect to the large subunit, and tRNA appeared to be bound in the hybrid P/E state () [10
]. This finding prompted the proposal that translocation is driven by a ratchet-like mechanism that is coupled to intersubunit rotation. In a critical test of this model, introduction of an intersubunit disulfide bridge between protein S6 on the 30S subunit and L2 on the 50S subunit was found to specifically block translocation, showing that intersubunit movement is indeed required for translocation [13
]. The axis of rotation was localized to the vicinity of intersubunit bridge 3 (residues 1483-1486 of h44 of 16S RNA and 1948-1949 of h71 of 23S rRNA). Accordingly, intersubunit bridges located near the rotation axis (B2a-c, B3, B5 and B7a), appear to be essentially maintained during intersubunit rotation, while bridges located at the extremities of the subunits (such as B1a-b, B7b and B8) are disrupted or rearranged.
Recently, intersubunit rotation has been directly observed in solution using FRET. Binding of EF-G was found to cause counterclockwise rotation of the small subunit in ribosomes containing fluorophores attached to proteins S6 (or S11) in the 30S subunit and L9 in the 50S subunit [14
]. By combining chemical probing and FRET studies, it was shown that the EF-G-induced rotation corresponds to formation of the hybrid state characterized in the early chemical probing studies, and can occur in the absence of EF-G under conditions that favor spontaneous hybrid state formation [14
]. Thus, the hybrid-state and ratchet models have converged on a common unified mechanism for translocation ().
A shortcoming of ensemble FRET experiments is that the behavior of individual molecules is masked by averaging. This problem is overcome by single-molecule FRET (smFRET) experiments. Using smFRET, fluorescently-labeled tRNAs bound to pre-translocation ribosomes were observed to move spontaneously relative to one another, interpreted as fluctuations between the classical and hybrid states [16
]. This was shown directly in smFRET experiments using fluorescently-labeled ribosomal subunits, in which pre-translocation ribosomes containing deacylated tRNA in the P site were seen to fluctuate spontaneously between two rotational conformations corresponding to the classical and hybrid states [19
]. In contrast, post-translocation ribosomes, containing peptidyl-tRNA in the P site were fixed predominantly in the classical, non-rotated state. Analysis of equilibria between the two intersubunit conformations, based on FRET and chemical probing data, shows that stabilization of the rotated, hybrid state is influenced both by movement of the acceptor stem of deacylated tRNA into the 50S E site and by binding of EF-G [19
Binding of initiation factor IF2 [21
], release factor RF3 [14
] or ribosome recycling factor RRF [24
] also causes movement into the rotated, hybrid state, extending the involvement of intersubunit rotation to the initiation and termination phases of protein synthesis. Moreover, counterclockwise rotation of the small subunit was also seen in cryo-EM reconstructions of EF-2-containing complexes of the eukaryotic ribosome [25
]. Thus, intersubunit movement and the hybrid-state intermediate appear to be universal features of translation that account for conservation of the two-subunit organization of ribosomes among all branches of life.