## 3 Structural Snapshots of tRNA Conformation Plasticity during Translation
### Translocation  
The rapid peptide transfer occurs after the accommodation of the incoming tRNA in the A site [149, 150, 151, 152], leading to the formation of the so-called pre-translocational complex, ready to translocate the tRNA-mRNA complex by one codon in the 5' to 3' direction once unlocked [153, 154]. First, the acceptor stems of the A and P tRNAs move with respect to the large subunit, generating the hybrid A/P and P/E configurations [17]. Kinetic data has shown that formation of the P/E hybrid configuration precedes the A/P state formation [31, 41]. This tRNA reconfiguration is linked to the rotation of the 30S relative to the 50S along with the L1 stalk reconfiguration (first detected in EF-G bearing complexes, see [155, 156]), and it is spontaneously formed in pre-translocational complexes as demonstrated by kinetic and FRET [29, 30, 34, 39, 157, 158, 159], and cryoEM studies of prokaryotic [160, 161] as well as eukaryotic [162, 163] complexes. X-ray data initially showed that the ribosome wraps intermediate rotated structures [164]. Later on, several intermediates, which include different tRNA configurations, have been described by cryoEM as well [165, 166, 167, 168] (Figure 7), and the spatial and temporal relation between these transition state configurations and 30S subunit and L1 rearrangements computationally analyzed [61, 62, 63]. Complete translocation of the tRNAs from the A and P sites to the P and E sites respectively requires EF-G (or its eukaryotic counterpart eEF2, e.g., see [169, 170]) and is coupled to the rotation of the head of the small subunit [171, 172, 173, 174]. The factor, which undergoes a drastic structural rearrangement from a compact to an elongated conformation once bound to the ribosome [175], but before the hydrolysis of GTP [176], initiates mRNA-tRNA translocation by breaking the interactions established between the cognate codon-anticodon duplex and the decoding center [169, 177, 178, 179]. Kinetic data have shown that the hydrolysis of GTP is very rapid and precedes translocation [31, 180, 181, 182, 183]. On the other hand, it has been demonstrated that EF-G can bind to the ribosome in either the classical or hybrid state to promote translocation [42, 44, 184], but favors the hybrid configuration [30, 185, 186]. It also has been shown that the final translocation of the tRNA-mRNA complex is coupled with the reverse rotation from the hybrid state to the classical state [187]. Figure 7: Comparison of translocation intermediates obtained by cryoEM and particle sorting. **(a–f)** Maximum likelihood methods were applied to a dataset of pre-translocational complexes in the absence of EF-G, obtaining an ensemble of substates that encompass a large conformational space. Out of the initial six classes, one subset (class 4) was further subdivided due to residual heterogeneity (class 1 is not shown as it was deemed to be artifactual due to bias in particle orientations). Class 2 **(a)** and class 4A **(c)** correspond to the classic A/A and P/P tRNA configuration, while class 3 **(b)** corresponds to ribosomes bearing a single tRNA configuration (P/P). Classes 5 **(e)** and 6 **(f)** are similar to each other, and to the previously characterized A/P and P/E hybrid configurations [160, 161]. Class 4B **(d)** represents a new intermediate in which the P-site tRNA’s elbow is halfway through its transition from classic to hybrid state, a configuration that is coupled to an intermediate intersubunit rotation and L1 stalk movement; **(g)** Stereo-view of the superposition of A- and P-site tRNAs from class 2 (classic state tRNAs, in magenta and green), class 4B (new intermediates, in olive) and class 6 (hybrid state tRNAs, in gray), aligned with respect to the 70S ribosomes. (Figure adapted from [168], Copyright 2012 Proc. Natl. Acad. Sci. USA). Recent high resolution structures of EF-G bound ribosomal complexes have shed light on the process [103, 172, 177, 190, 191, 192, 193, 194]. One of the most relevant structures is the crystal structure of a _T.