Mapping important nucleotides in the peptidyl transferase centre of 23 S rRNA using a random mutagenesis approach

Random mutations were generated in the lower half of the peptidyl transferase loop in domain V of 23 S rRNA from Escherichia coli using a polymerase chain reaction (PCR) approach, a rapid procedure for identifying mutants and a plasmid-based expression system. The effects of 21 single-site mutations, at 18 different positions, on cell growth, mutant rRNA incorporation into ribosomes and peptidyl transferase activity of the mutant ribosomes were analysed. The general importance of the whole region for the peptidyl transferase centre was emphasized by the finding that 14 of the mutants were sick, or very sick, when ribosomes containing chromosomal-encoded 23 S rRNA were inhibited by erythromycin, and all except one of these exhibited low levels of peptidyl transferase activity in their mutated ribosomes. Two mutations, psi 2580-->C and U2584-->G that both yielded inactive ribosomes were assigned to the donor substrate binding site and a possible base-pairing interaction between the 3'-terminal sequence of the peptidyl-tRNA and the sequence psi/U-G-G2582, that is conserved in all the non-mitochondrial 23 S-like rRNA sequences, is proposed. Three sites that have been implicated in aminoacyl-tRNA binding were mutated: mutant m6A2503G yielded inactive ribosomes, while ribosomes from mutants Um2552A/C and U2555C yielded low and normal activities, respectively. Three mutants, U2528C, G2550A and A2565U, provide evidence for conformational rearrangements occurring in the peptidyl transferase centre which may be affected by the subunit-subunit interaction. Other mutants which yielded ribosomes that were seriously defective in peptidyl transferase activity were U2493A, U2493C, A2497G, A2530G, G2557A and A2589G.