We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.

Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.

The widely used antibiotic spectinomycin inhibits bacterial protein synthesis by blocking translocation of messenger RNA and transfer RNAs on the ribosome. Here, we show that in crystals of the Escherichia coli 70S ribosome spectinomycin binding traps a distinct swiveling state of the head domain of the small ribosomal subunit. Spectinomycin also alters the rate and completeness of reverse translocation in vitro. These structural and biochemical data indicate that in solution spectinomycin sterically blocks swiveling of the head domain of the small ribosomal subunit and thereby disrupts the translocation cycle.Spectinomycin, a broad-spectrum antibiotic that selectively targets bacterial ribosomes (1-3), remains important in clinical and veterinary use, as it induces only low levels of bacterial resistance (4-6). Despite decades of worldwide use of the drug, the molecular mechanism by which spectinomycin inhibits translation remains unclear.Spectinomycin, an aminocyclitol constituted from two glucose moieties (7) (Figure 1, panel a), inhibits translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) on the ribosome (8-10), the step that follows the formation of each peptide bond. Translocation, catalyzed by the GTPase elongation factor G (EF-G), involves multiple large conformational * Corresponding author, jcate@lbl.gov.. Supporting Information Available:This material is available free of charge via the Internet.Accession Codes: The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 2QOU, 2QOV, 2QOW, and 2QOX (70S ribosome in complex with spectinomycin), and 2QOY, 2QOZ, 2QP0, and 2QP1 (70S ribosome in complex with spectinomycin and neomycin). HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript changes in the ribosome (9,(11)(12)(13)(14)(15). Translocation is thought to begin with a ratchet-like motion of the small (30S) ribosomal subunit relative to the large (50S) ribosomal subunit (11,15,16), followed by swiveling of the head of the small subunit (12, 13) and "unlocking" (opening) of the tRNA binding groove to allow peptidyl (P-site) tRNA to pass into the Exit (E) site (13,14,17,18). These conformational changes lead to discrete movements of bound tRNAs, where the tRNAs move first with respect to the 50S subunit into hybrid binding states and then move with respect to the 30S subunit (19). Unlike most of the translocation inhibitors, spectinomycin slows but does not completely abolish multiple-turnover translocation (9). It also does not induce mRNA miscoding (20, 21).The binding site for spectinomycin, in RNA helix 34 (h34) of the head domain of the 30S subunit, resides near the single ribosomal RNA (rRNA) helix that connects the head domain to the rest of the small subunit (i.e., neck of the 30S subunit) and faces the mRNA binding groove (22-26) (Figure 1, panels b and c; Supplementary Figure 1). Biochemical data (9,24,25,27) and the structure of spectinomycin bound to the 30S ribosomal subunit (26) suggested ...