Crystallographic and mutational studies of the antibiotic specificity of the ribosome

Many inhibitors of the large ribosomal subunit block the activity of the ribosome by binding at the peptidyl transferase center (PTC), where peptide bonds form. Most of these inhibitors bind at the A-site and interfere with the binding of the amino acid side chains of the incoming A-site tRNAs. Despite the high level of conservation in that binding site, some inhibitors are orders of magnitude more active against eubacterial ribosomes than eukaryotic ribosomes, while others have the opposite specificity, or no specificity at all. In this study, the role of the bases in the 23S rRNA and the residues of ribosomal proteins that are involved in controlling the species specificity of ribosomal antibiotics have been investigated using mutational and X-ray crystallographic techniques.;Anisomycin is a eukaryote-specific inhibitor that interferes with the binding of the A-site tRNA to the PTC. Spontaneous and site-directed mutations in 23S and L3 have been obtained in Haloarcula marismortui (H.marismortui) that confer anisomycin resistance. The structures of large ribosomal subunits of H.marismortui containing these mutations have been determined by X-ray crystallography. These mutations confer resistance in two different ways: (1) by blocking specific drug-ribosome interactions, or (2) by stabilizing the apo-conformation of the A-site cleft relative to its drug bound conformation through long range interactions. Thus, the analysis of these structures has revealed that there are several conformational states available to the A-site cleft, and that the equilibrium between the conformational states can be modulated by sequences that are away from them.;The conclusions obtained from the studies done with anisomycin are supported by the structures that were obtained from the complexes the large ribosomal subunit of H.marismortui forms with eubacteria specific A-site cleft antibiotic tiamulin, and eukaryote-specific A-site cleft inhibitors homoharringtonine and bruceantine. Comparison of these structures with the structure of tiamulin bound to eubacterial large ribosomal subunit supports the conclusion that the specificity of the A-site can be modulated by bases or residues that are a nucleotide or two away from them. The species specificity of the A-site appears to be controlled by the interactions the antibiotics make or fail to make with a single nucleotide U2504. The position of U2504 is controlled by its interactions with neighboring nucleotides, whose identities vary among the different kingdoms of life.;In the course of this work, crystal structures of complexes of H.marismortui large ribosomal subunit with triacetyloleandomycin and myacalamide-A were obtained. Triacetyloleandomycin, a eubacteria specific macrolide, binds at the entrance of the peptide tunnel in the mutated ribosomes (G2058A) of H.ma. Like Erythromycin, its specificity for ribosomes is determined by the identity of the base G2058. Eukaryote-specific ribosomal inhibitor mycalamide-A binds at the E-site of the large ribosomal subunit and makes contacts with both 23S and L44e. The crystal structure reveals that its specificity is determined by the interactions it makes with L44e, a ribosomal protein found only in archaea and eukaryotes.