RECOGNITION DETERMINANTS FOR PROTEINS AND ANTIBIOTICS WITHIN 23S RIBOSOMAL-RNA

Ribosomal RNAs fold into phylogenetically conserved secondary and tert iary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural el ements that interact with antibiotic and protein ligands. Using a comb ination of molecular genetic and biochemical probing techniques, we ha ve concentrated on regions of the rRNA that are connected with specifi c functions. These are located in different domains within the 23S rRN A and include the ribosomal GTPase-associated center in domain II, whi ch contains the binding sites for r-proteins L10 .(L12)(4) and L11 and is inhibited by interaction with the antibiotic thiostrepton. The pep tidyltransferase center within domain V is inhibited by macrolide, lin cosamide, and streptogramin B antibiotics, which interact with the rRN A around nucleotide A2058. Drug resistance is conferred by mutations h ere and by modification of A2058 by ErmE methyltransferase. ErmE recog nizes a conserved motif displayed in the primary and secondary structu re of the peptidyl transferase loop. Within domain VI of the rRNA, the alpha-sarcin stem-loop is associated with elongation factor binding a nd is the target site for ribotoxins including the N-glycosidase ribos ome-inactivating proteins ricin and pokeweed antiviral protein (PAP). The orientations of the 23S rRNA domains are constrained by tertiary i nteractions, including a pseudoknot in domain II and long-range base p airings in the center of the molecule that bring domains II and V clos er together. The phenotypic effects of mutations in these regions have been investigated by expressing 23S rRNA from plasmids. Allele-specif ic priming sites have been introduced close to these structures in the rRNA to enable us to study the molecular events there.