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.

Mycobacterium abscessus is a fast-growing, multidrug-resistant organism that has emerged as a clinically significant pathogen in cystic fibrosis (CF) patients. The intrinsic resistance of M. abscessus to most commonly available antibiotics seriously restricts chemotherapeutic options. Herein, we report the potent activity of a series of indolecarboxamides against M. abscessus. The lead compounds, 6 and 12, exhibited strong activity in vitro against a wide panel of M. abscessus isolates and in infected macrophages. High resistance levels to the indolecarboxamides appear to be associated with an A309P mutation in the mycolic acid transporter MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis remained unaffected, the indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan. Our data introduce a hereto unexploited chemical structure class active against M. abscessus infections with promising translational development possibilities for the treatment of CF patients.

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.

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.