The process of mRNA–tRNA translocation

Frank et al. 10.1073/pnas.0708517104.

Supporting Information

Files in this Data Supplement:

SI Figure 6
SI Movie 1
SI Figure 7
SI Figure 8

SI Figure 6

Fig. 6. Flexible fitting of the E. coli 70S ribosomal complexes using RSRef. (a) Overall fitting of a pretranslocational complex. (b and c) Comparison of the atomic models of the rRNAs obtained by fitting of EM density maps of the pretranslocational and EF-G-bound, ratcheted complexes (1). The real-space refinement fitting was performed by using RSRef (2) and TNT (3). The 3.5-Å crystal structure of the E. coli ribosome (4) was fitted into the cryo-EM maps of the PRE ribosome and the EF-G•GDPNP-bound, ratcheted ribosome (1). A multi-rigid body refinement scheme was employed as previously described (5). Briefly, ribosomal RNA was divided into rigid bodies based on secondary structure, and ribosomal proteins were each treated as a single rigid body. In all, 157 pieces were subjected to real-space refinement for each ribosomal complex.

1. Valle M, Zavialov A, Sengupta J, Rawat U, Ehrenberg M, Frank J (2003) Cell 114:123-134.

2. Chapman MS (1995) Acta Crystallogr A 51:69-80.

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4. Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A, Holton JM, Cate JH (2005) Science 310:827-834.

5. Gao H, Sengupta J, Valle M, Korostelev A, Eswar N, Stagg SM, VanRoey P, Agrawal RK, Harvey SC, Sali A, Chapman MS, Frank J (2003) Cell 113:789-801.

SI Figure 7

Fig. 7. Binding of ribosomal GTPases induce ratcheting of the small subunit. (Top) The pretranslocational ribosome with no factors bound and P-site tRNA reveals one conformation in the cryo-EM reconstruction. (Middle) The binding of EF-G, RF3, IF2, and RRF induces a ratcheting of the small ribosomal subunit with respect to the large ribosomal subunit. (Bottom) When the large subunits of the different reconstructions are aligned, the small subunit of the factor-bound complex (pink volume) is seen to be rotated counterclockwise when overlaid with the small subunit of the pretranslocational complex (yellow volume) and viewed from the solvent side of the ribosome. Of the factors, IF2, EF-G, and RF3 are all GTPases. RRF is not a GTPase but requires the GTPase activity of EF-G to separate and recycle the ribosomal subunits (1). The coloring of segmented volumes is the same as in Fig. 2.

1. Gao N, Zavialov AV, Li W, Sengupta J, Valle M, Gursky RP, Ehrenberg M, Frank J (2005) Mol Cell 18:663-674.

SI Figure 8

Fig. 8. The various conformations of EF-G. The conformational changes observed in EF-G involve a hinge-like movement of the C-terminal domains (III, IV, and V) with respect to the N-terminal domains (I, II, and G′). (Middle) The domains of EF-G are color-coded, and the direction of tRNA movement from the A site to the P site on the ribosome is depicted. (Top) The conformation of EF-G•GTP in solution (red) is taken from the x-ray crystal structure of the EF-G homolog EF-G-2 in complex with GTP (1). Once EF-G•GTP binds to the ribosome (yellow), the factor undergoes gross conformational changes that yield a shift in the tip of domain IV of EF-G by ≈27 Å. The large conformational changes in EF-G, upon binding to the ribosome, likely facilitate some movement of the tRNAs in the direction of translocation while stabilizing the tRNAs in the hybrid positions and the ribosome in the ratcheted orientation. The EF-G•GTP•ribosome conformation was determined by cryo-EM using the nonhydrolyzable GTP analog GDPNP (2). (Right) The conformational changes in ribosome-bound EF-G, upon GTP hydrolysis, are likely similar to those described for eEF2•80S complexes (3). In this scenario, the GDPNP-bound conformation is again shown in yellow and the GDP-bound conformation in blue (both are bound to the ribosome). A comparison of the complexes before and after GTP hydrolysis revealed a small shift in domains I, II, and G′ toward the GAC of the ribosome, a reorganization of the switch 1 loop, and an ≈6-Å shift in domain IV toward the decoding center of the ribosome. This 6-Å shift in domain IV likely severs the connection between the decoding center in the body of the small subunit and the A-site mRNA-tRNA duplex bound to the head of the small subunit, so that movement of the mRNA-tRNA complex can occur via a head rotation of the small subunit (3). (Bottom) After the head rotation of the small subunit, along with translocation of the anticodon stem-loops of tRNAs and the mRNA chain, EF-G•GDP dissociates from the ribosome. Conformational changes of EF-G•GDP from the ribosome-bound conformation (blue), determined by cryo-EM, to the solution structure (pink), determined by x-ray crystallography, include a movement at the tip of domain IV by ≈43 Å. This conformational change in EF-G likely contributes to its dissociation, which would then allow the ribosome to back-ratchet, resulting in a posttranslocational ribosome. Accommodation of aa-tRNA into the A site of the posttranslocational ribosome then results in formation of the pretranslocational ribosome. (Left) A comparison of the solution structures, determined by x-ray crystallography, of EF-G with different nucleotides bound. A comparison of the GDP-bound conformation (pink) of EF-G (4) with the GTP-bound conformation (red) of EF-G-2 (1) reveals a shift at the tip of domain IV of the factor by ≈25 Å. Binding of the triphosphate guanosine also reveals an ordered switch 1 loop and a different conformation of the switch 2 loop. The sum of these conformational changes results in a much higher affinity of the pretranslocational ribosome for the GTP-bound conformation of EF-G.

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SI Movie 1

Movie 1. Animation of the ribosomal conformational change from the pretranslocational state to the ratcheted state. The quasi-atomic models were obtained by fitting the x-ray structure (1) into EM density maps of the pretranslocational and EF-G-bound, ratcheted complexes (2), using real-space refinement (3, 4).

1. Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A, Holton JM, Cate JH (2005) Science 310:827-834.

2. Valle M, Zavialov A, Sengupta J, Rawat U, Ehrenberg M, Frank J (2003) Cell 114:123-134.

3. Chapman MS (1995) Acta Crystallogr A 51:69-80.

4. Fabiola F, Chapman MS (2005) Structure (London) 13:389-400.