Energy barriers and driving forces in tRNA translocation through the ribosome (original) (raw)

2013, Nature Structural & Molecular Biology

nature structural & molecular biology advance online publication a r t i c l e s Ribosomes are molecular machines that synthesize proteins from aminoacyl tRNAs, using mRNA as template. After formation of a peptide bond, the two tRNAs bound to the aminoacyl (A) and peptidyl (P) sites on the small (30S) and large (50S) ribosomal subunits translocate by more than 7 nm to the P and exit (E) sites, respectively, while the next mRNA codon moves into the A site . During translocation, tRNAs move on the 50S subunit into the hybrid A/P and P/E positions 1 with a concomitant rotation of the 30S subunit relative to the 50S subunit 2-4 . The rate-limiting step of translocation is the displacement of the codon-anticodon complexes on the 30S subunit; this, followed by the reversal of the subunit rotation, yields the post-translocation complex. Translocation is promoted by elongation factor G (EF-G) and is driven by GTP hydrolysis. In the absence of the factor, spontaneous, thermally driven tRNA translocation can occur 5-8 , and this seems to involve the same intersubunit interactions that occur in the presence of EF-G 9 . Spontaneous translocation is an equilibrium process, in which the tRNAs make rapid, spontaneous excursions in both forward and backward directions 5,6,10 . Preferential directionality is determined by the affinities of the tRNAs for their respective binding sites 5,6 . The process of translocation entails fluctuations of tRNAs 4,11-14 and of the components of the 50S subunit such as the L1 stalk 3,15-17 . A recent cryo-EM work revealed a large number of different conformational states for spontaneous, thermally driven tRNA movement through the ribosome 10 . However, precisely how the thermal fluctuations of tRNAs and of parts of the ribosome cooperatively drive the tRNA movement is unclear. It is also unclear whether and how synchronous movements-such as those involving intersubunit rotations, the L1 stalk and tRNA fMet -are coupled to one another. Furthermore, it is unknown how efficient tRNA handover from one binding site to another is achieved, despite the considerable structural changes along the translocation path. To address these questions, we combined data from X-ray crystallography and singleparticle cryo-EM with molecular dynamics (MD) simulations.