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.

SummaryStructures of the E. coli 70S ribosome show how the large and small subunits rotate to facilitate protein synthesis.Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo form (3.5 Å resolution) or with one (4.0 Å res) or two (4.0 Å res) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.Protein biosynthesis requires many large-scale rearrangements in the ribosome as each amino acid is added to a growing polypeptide chain. Positioning of tRNA on the ribosome is proposed to occur through a ratcheting mechanism. Central to this mechanism is a rotation of the small ribosomal subunit relative to the large subunit (Fig. 1A) (1,2) that occurs in all stages of translation-initiation, elongation, termination, and ribosome recycling (1)-and is targeted by clinically useful antibiotics (3,4). For example after each peptide bond is formed, an ~8°i ntersubunit rotation results in tRNAs bound in the aminoacyl-tRNA and peptidyl-tRNA binding sites (A site and P site, respectively) moving into the P site and exit-tRNA site (E site) on the large ribosomal subunit (Fig. 1B). From this hybrid state of tRNA binding (Fig. 1B) (1,5), the tRNAs are then translocated to the P site and E site on the small subunit.In addition to intersubunit rotation, ratcheting also involves a nearly orthogonal rotation of the head domain of the small ribosomal subunit (Fig. 1C) that plays a role in controlling the position of tRNAs within the ribosome (1,6,7). As with intersubunit rotation, movement of the head domain is a target for clinically useful antibiotics (8). Swiveling of the head domain relative to the body of the small subunit may also be required for the intrinsic helicase activity of the ribosome in unwinding secondary structure in mRNA (8,9). Rotations of up to 14° allow the head domain to change its position by 20 Å or more at the ribosomal subunit interface, or the width of a tRNA substrate (7). The molecular basis for how the ribosomal subunits rotate with respect to each other while remaining stably associated remains unknown (1,10). Furthermore, the precise timing of movements of the small subunit head domain during ra...