MUTATIONAL ANALYSIS OF THE DONOR SUBSTRATE-BINDING SITE OF THE RIBOSOMAL PEPTIDYLTRANSFERASE CENTER

Previous experiments have shown that the top of helix 90 of 23S rRNA i s highly important for the ribosomal peptidyltransferase activity and might be part of the donor (P) site. Developing on these studies, muta tions in the 23S rRNA at the highly conserved positions G2505, G2582, and G2583 were investigated. None of the mutations affected assembly, subunit association, or the capacity of tRNA binding to A and P sites. A ''selective transpeptidation assay'' revealed that the mutations sp ecifically impaired peptide bond formation. Results with a modified '' fragment'' assay using the minimal donor substrate pA-fMet are consist ent with a model where the nucleotides Psi GG2582 form a binding pocke t for C75 of the tRNA.