RIBOSOMAL-PROTEIN L27 PARTICIPATES IN BOTH 50-S-SUBUNIT ASSEMBLY AND THE PEPTIDYL TRANSFERASE REACTION

Protein L27 has been implicated as a constituent of the peptidyl trans ferase center of the Escherichia coli 50 S ribosomal subunit by a vari ety of experimental observations. To define better the functional role ! of this protein, we constructed a strain in which the rpmA gene, whi ch encodes L27, was replaced by a kanamycin resistance marker. The del etion mutant grows five to six times slower than the wild-type parent and is both cold-and temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Ana lysis of ribosomes from the L27-lacking strain revealed deficiencies i n both the assembly and activity of the 50 S ribosomal subunits. Altho ugh functional 50 S subunits are formed in the mutant, an assembly ''b ottleneck'' was evidenced by the accumulation of a prominent 40 S prec ursor to the 50 S subunit which was deficient in proteins Lie, L20, an d L21, as web as L27. In addition, the peptidyl transferase activity o f 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes, Ribosomes lack ing L27 were found to be impaired in the enzymatic binding of Phe-tRNA (Phe) to the A site, although the interaction of N-acetyl-Phe-tRNA(Phe ) with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper plac ement of the acceptor end of the A-site tRNA at the peptidyl transfera se center.