Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3

Abstract

The small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. We analyzed by X-ray crystallography the structures of three different complexes of the small ribosomal subunit of Thermus thermophilus with the A-site inhibitor tetracycline, the universal initiation inhibitor edeine and the C-terminal domain of the translation initiation factor IF3. The crystal structure analysis of the complex with tetracycline revealed the functionally important site responsible for the blockage of the A-site. Five additional tetracycline sites resolve most of the controversial biochemical data on the location of tetracycline. The interaction of edeine with the small subunit indicates its role in inhibiting initiation and shows its involvement with P-site tRNA. The location of the C-terminal domain of IF3, at the solvent side of the platform, sheds light on the formation of the initiation complex, and implies that the anti-association activity of IF3 is due to its influence on the conformational dynamics of the small ribosomal subunit.

Fig. 1. (A) Stereo view of the 3.2 Å structure of the 30S ribosomal subunit. The binding sites of tetracycline and edeine are indicated. Ribosomal proteins interacting with tetracycline have been colored and labeled. (B and C) Stereo views of the electron density map of two regions of the 3.2 Å structure, corresponding to helix 16 (H16) and ribosomal protein S16, respectively.

Fig. 2. Details of the binding sites of tetracycline and edeine. All images show the σA weighted difference density at contour levels ranging from 3.2 to 2.2σ for tetracycline, and 3.2σ for both edeine and the RNA base pair induced upon its binding. (A) Stereo view of Tet-1. (B) Mono view of Tet-2. (C) Mono view of Tet-3. (D) Stereo view of Tet-4 and -6. (E) Left: stereo view of Tet-5. Right: chemical structure of tetracycline. (F) Left: stereo view of the binding site of edeine. Right: chemical structure of edeine.

Fig. 3. Binding of IF3 to the small subunit. (A) The model of the complete 30S–IF3C complex (IF3C in red) and the docking of IF3N (blue) is shown. (B) Two regions of the σA weighted difference map contoured at 3σ. (C) Mono view of the 30S particle (on the left) having the same orientation as the stereo view of the model of the 30S–IF3 complex (on the right) in the vicinity of IF3C. Parts of the 30S structure not involved in the interactions with IF3 are omitted for clarity. (D) Secondary structure of IF3C. Residues of IF3C interacting with different RNA helices and ribosomal proteins are highlighted. Nomenclature of the secondary structure is based on Biou et al. (1995). Above the secondary structure diagram are the rRNA regions and below the diagram are the proteins that interact with IF3C.