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.

After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosomerecycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by Ϸ13 Å) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA. R ibosomes are responsible for translating genetic information carried by mRNAs into specific sequences of amino acids. Translation on the ribosome comprises of four main steps: (i) initiation, (ii) elongation, (iii) termination, and (iv) recycling. Recent advancements in structural studies of the translational machinery have helped elucidate binding positions and functions of various translational factors involved in various stages of initiation (1, 2), elongation (3, 4), and termination (5-7). The fourth step of translation requires binding of a dedicated protein factor, the ribosome-recycling factor (RRF), which in conjunction with elongation factor G (EF-G) helps removing the mRNA and last deacylated tRNA from the ribosome (see ref. 8).Atomic structures of RRF determined from five different species, including Escherichia coli, show that it is comprised of two structural domains: domain I, consisting of three long ␣-helix bundles, and the smaller domain II, which is an ␣͞␤ motif (9-13). Different orientations of domain II in these structures have been attributed to interdomain flexibility, which is thought to be necessary for RRF to function on the ribosome (12).The overall match in dimensions between RRF and tRNA (9) prompted the proposal of structural and functional molecular mimicry between the two molecules (9). In a recent study (14) using the hydroxyl radical probing (HRP) method, the orientation of RRF on the ribosome was derived. This study did not support the idea of direct molecular mimicry of tRNA by RRF, because the derived binding position of RRF was quite different from that one would expect based on structural mimicry. The inferred bin...

Ribosome recycling factor (RRF), together with elongation factor G (EF-G), catalyzes recycling of ribosomes after one round of protein synthesis. The crystal structure of RRF was determined at 2.55 angstrom resolution. The protein has an unusual fold where domain I is a long three-helix bundle and domain II is a three-layer beta/alpha/beta sandwich. The molecule superimposes almost perfectly with a transfer RNA (tRNA) except that the amino acid-binding 3' end is missing. The mimicry suggests that RRF interacts with the posttermination ribosomal complex in a similar manner to a tRNA, leading to disassembly of the complex. The structural arrangement of this mimicry is entirely different from that of other cases of less pronounced mimicry of tRNA so far described.