MAPPING IMPORTANT NUCLEOTIDES IN THE PEPTIDYL TRANSFERASE CENTER OF 23-S RIBOSOMAL-RNA USING A RANDOM MUTAGENESIS APPROACH

Random mutations were generated in the lower half of the peptidyl tran sferase loop in domain V of 23 S rRNA from Escheuichin coli using a po lymerase chain reaction (PCR) approach, a rapid procedure for identify ing mutants and a plasmid-based expression system. The effects of 21 s ingle-site mutations, at 18 different positions, on cell growth, mutan t rRNA incorporation into ribosomes and peptidyl tranferase activity o f the mutant ribosomes were analysed. The general importance of the wh ole region for the peptidyl transferase centre was emphasized by the f inding that 14 of the mutants were sick, or very sick, when ribosomes containing chromosomal-encoded 23 S rRNA were inhibited by erythromyci n, and all except one of these exhibited low levels of peptidyl transf erase 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 intera ction between the 3'-terminal sequence of the peptidyl-tRNA and the se quence Psi/U-G-G(2582), that is conserved in all the non-mitochondrial 23 S-like rRNA sequences, is proposed. Three sites that have been imp licated in aminoacyl-tRNA binding were mutated: mutant m(6)A2503G yiel ded inactive ribosomes, while ribosomes from mutants Um2552A/C and U25 55C yielded low and normal activities, respectively. Three mutants, U2 528C, G2550A and A2565U, provide evidence for conformational rearrange ments occurring in the peptidyl transferase centre which may be affect ed by the subunit-subunit interaction. Other mutants which yielded rib osomes that were seriously defective in peptidyl transferase activity were U2493A, U2493C, A2497G, A2530G, G2557A and A2589G.