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 chemokine receptor CXCR4 is critical for many biological functions, such as B-cell lymphopoiesis, regulation of neuronal cell migration, and vascular development (1-3). In addition, CXCR4 together with another chemokine receptor CCR5 are two principal co-receptors for the cellular entry of the human immunodeficiency virus type 1 (HIV-1) 1 (4 -7). The stromal cell-derived factor-1 (SDF-1␣) is the only known natural ligand of CXCR4 and plays important roles in migration, proliferation, and differentiation of leukocytes (8, 9). The viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus 8 (10) is an antagonistic chemokine ligand of CXCR4 (11, 12). vMIP-II also interacts with other chemokine receptors such as CCR5 and CCR3 and inhibits HIV-1 entry mediated by these co-receptors.CXCR4 and other chemokine receptors belong to the superfamily of seven transmembrane G-protein-coupled receptors (GPCRs) (13). These membrane proteins transmit signals from extracellular ligands to intracellular biological pathways via heterotrimeric G-proteins and have been a major class of therapeutic targets for a wide variety of human diseases (14). As such, characterizing the mechanism of biological recognition between these receptors and their ligands is essential for understanding the physiological or pathological processes that they mediate and devising novel strategies for clinical intervention. For CXCR4, studies have been carried out by a number of laboratories using chimeric chemokine receptors and site-specific mutants to study multiple domains of CXCR4 that are important for interacting with chemokine ligands and HIV-1 (15-23). However, because there is no high resolution crystal structure available for CXCR4 (or any other chemokine receptor) alone or complexed with ligands, the structural and biochemical basis of ligand binding and signaling through these important membrane receptors remains poorly understood.To further define the structure-function relationship of the chemokine receptor-ligand interaction, theoretical computer modeling and site-directed mutagenesis were combined to predict plausible structural models for chemokine receptors and their complexes with ligands, such as interleukin-8 receptor ␤ (24) and CCR5 (25,26). Structural models of CXCR4 and its complex with ligands were also proposed (27, 28). Complementary to modeling and mutational analyses of the receptors,

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The threefactor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.

After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mRNA and deacylated tRNA from the PoTC, are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling.

The ribosome-recycling factor (RRF) and elongation factor-G (EF-G) disassemble the 70S post-termination complex (PoTC) into mRNA, tRNA, and two ribosomal subunits. We have determined cryo-electron microscopic structures of the PoTC . RRF complex, with and without EF-G. We find that domain II of RRF initially interacts with universally conserved residues of the 23S rRNA helices 43 and 95, and protein L11 within the 50S ribosomal subunit. Upon EF-G binding, both RRF and tRNA are driven towards the tRNA-exit (E) site, with a large rotational movement of domain II of RRF towards the 30S ribosomal subunit. During this intermediate step of the recycling process, domain II of RRF and domain IV of EF-G adopt hitherto unknown conformations. Furthermore, binding of EF-G to the PoTC . RRF complex reverts the ribosome from ratcheted to unratcheted state. These results suggest that (i) the ribosomal intersubunit reorganizations upon RRF binding and subsequent EF-G binding could be instrumental in destabilizing the PoTC and (ii) the modes of action of EF-G during tRNA translocation and ribosome-recycling steps are markedly different.