Fine structure of the peptidyl transferase centre on 23 S-like rRNAs deduced from chemical probing of antibiotic-ribosome complexes.

Ribosomal binding sites were investigated for the diverse group of antibiotics: anisomycin, anthelmycin, blasticidin S, bruceantin, carbomycin, chloramphenicol, griseoviridin, narciclasine, T2 toxin, tylosin and virginiamycin M1 all of which are considered to inhibit the peptidyl transferase reaction by different mechanisms. The drugs also exhibit differing degrees of specificity for bacterial, archaeal and eukaryotic ribosomes despite a high level of conservation of sequence and secondary structure at the peptidyl transferase centre of the 23 S-like rRNAs. The drug binding sites were characterized by incubating each antibiotic with ribosomes from a bacterium, an archaeon and a eukaryote and chemically probing the 23 S-like rRNA. The complexity of the changes in reactivity ranged from one or two nucleotides (anthelmycin, narciclasine) to eight or nine (virginiamycin M1) and it was inferred, at least for those drugs producing complex changes, that they induce, and stabilize, a particular functional conformer in the peptidyl transferase centre. The results were correlated with literature data on both ribosomal ligand binding and the putative inhibitory mechanisms of the drugs, and the following inferences are made concerning the fine structure of the peptidyl transferase centre. (1) An irregular secondary structural motif, which includes unpaired A2439 (Escherichia coli numbering), lies close to the catalytic centre; (2) nucleotides A2451 and C2452 contribute to a site for the binding of the side chains of aromatic amino acids; (3) the P-substrate site encompasses U2585, U2506 and, possibly, a site in domain IV (A1787), and (4) the sequence A2058 to A2062 and nucleotide U2609 contribute to, or modulate, the start of the peptide channel. No drug effects were found that could be directly attributed to an A-site and the possibility is raised that, if it exists, it consists mainly of ribosomal proteins. However, two drugs T2 toxin and virginiamycin M1 protected the only nucleotide in the peptidyl transferase loop region (C2394) associated with the E-site. Finally, it is proposed that the putative sub-sites are physically separated, that some drugs bind to more than one of them, and that they are conformationally interdependent.