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APPENDIX 4: Visualization of Elongation Factor Tu on the 70S E. coli Ribosome

Holger Stark, Marin van Heel, Marina Rodnina, Wolfgang Wintermeyer, Richard Brimacombe


The binding of aminoacyl-tRNAs to the ribosome is catalyzed by the elongation factor Tu (EF-Tu). The release of the ternary complex (EF-Tu/tRNA/GTP) from the ribosome usually occurs after GTP hydrolysis and can be prevented by the antibiotic kirromycin. Those ribosomes were embedded in amorphous ice by rapidly freezing the sample in its natural environment on a holey carbon grid and imaged in an electron microscope either at liquid helium or liquid nitrogen temperature. Three-dimensional structures of ribosomes in different functional states can then be calculated (Agrawal et al. 1996; Stark et al. 1997b) using advanced image processing procedures (van Heel et al. 1996). The structure of the kirromycin-stalled ribosome was determined to a resolution of 18 Å (Stark et al. 1997a). The kirromycin-stalled ribosome is shown here with the 50S subunit on the right and the 30S subunit on the left-hand side. The crystal structures of the ternary complex in red and the P-site tRNA in green were fitted to the cryo-EM density map. Domain 1 of the EF-Tu is bound to the L7/L12 stalk of the 50S subunit, whereas domain 2 is oriented toward the S12 region on the 30S subunit. The ternary complex is thus spanning the gap between the two subunits with the acceptor region of the tRNA reaching into the decoding center on the 30S subunit.

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