FINE-STRUCTURE OF THE PEPTIDYL TRANSFERASE CENTER ON 23 S-LIKE RIBOSOMAL-RNAS DEDUCED FROM CHEMICAL PROBING OF ANTIBIOTIC-RIBOSOME COMPLEXES

Ribosomal binding sites were investigated for the diverse group of ant ibiotics: anisomycin, anthelmycin, blasticidin S, bruceantin, carbomyc in, chloramphenicol, griseoviridin, narciclasine, T2 toxin, tylosin an d virginiamycin M(1) all of which are considered to inhibit the peptid yl transferase reaction by different mechanisms. The drugs also exhibi t differing degrees of specificity for bacterial, archaeal and eukaryo tic ribosomes despite a high level of conservation of sequence and sec ondary structure at the peptidyl transferase centre of the 23 S-like r RNAs. The drug binding sites were characterized by incubating each ant ibiotic with ribosomes from a bacterium, an archaeon and a eukaryote a nd chemically probing the 23 S-like rRNA. The complexity of the change s in reactivity ranged from one or two nucleotides (anthelmycin, narci clasine) to eight or nine (virginiamycin M(1)) and it was inferred, at least for those drugs producing complex changes, that they induce, an d stabilize, a particular functional conformer in the peptidyl transfe rase 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 stru cture of the peptidyl transferase centre. (1) An irregular secondary s tructural motif, which includes unpaired A2439 (Escherichia coil numbe ring), lies close to the catalytic centre; (2) nucleotides A2451 and C 2452 contribute to a site for the binding of the side chains of aromat ic 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 th e peptide channel. No drug effects were found that could be directly a ttributed to an A-site and the possibility is raised that, if it exist s, it consists mainly of ribosomal proteins. However, two drugs T2 tox in and virginiamycin M(1) protected the only nucleotide in the peptidy l 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 confo rmationally interdependent.