Search

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. [ABSTRACT FROM AUTHOR]

The tuberactinomycins are a family of basic cyclic peptides that exhibit potent antitubercular activity. These peptides are characterized by the presence of an amino acid with a 6-membered cyclic guanidine side chain (capreomycidine) and two or more 2,3-diaminopropionate residues. Viomycin (tuberactinomycin B) is a well-studied member of the family, was once prescribed for the treatment of tuberculosis, and has been shown to block translocation during protein biosynthesis. The gene cluster encoding viomycin biosynthesis was identified and cloned from Streptomyces vinaceus. The cluster was identified by screening genomic libraries with the viomycin phosphotransferase self-resistance gene (vph) and non-ribosomal peptide synthetase (NRPS) gene probes amplified from S. vinaceus genomic DNA. The viomycin cluster was localized to ca. 120 kb of contiguous DNA defined by four overlapping cosmid inserts. Each cosmid hybridized with one or more peptide synthetase gene probes and two also hybridized with vph. Confirmation that the cluster encoded viomycin biosynthesis was obtained from the disruption of two NRPS adenylation domains. Partial sequence analysis revealed an ORF (svox) predicted to encode a rare non-heme iron, α-ketoglutarate dependent oxygenase proposed to function in the oxidative cyclization of arginine to the capreomycidine residue. Insertional disruption of svox resulted in complete loss of viomycin production, confirming its involvement in the pathway. [Copyright &y& Elsevier]