Characterization of Mutations in Ribosomal Proteins L4 and L22 that confer Erythromycin Resistance in E. coli

Abstract

L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated "tentacles" that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. To learn more about the roles of the tentacles of L4 and L22 in ribosomal assembly and function, we isolated and characterized erythromycin resistant mutants in E. coli. All of the mutants grew slower than the parent and all showed reduced in vivo rates of peptide elongation and increased levels of precursor 23S rRNA. Sucrose gradient sedimentation analysis also showed that large insertions in L4 and L22 result in accumulation of abnormal ribosomal subunits. In addition, structural studies using chemical modification followed by primer extension reveal changes in rRNA structure due to large insertion mutations in L4 and L22, particularly at A2450 and A2451, key nucleotides in the peptidyl transferease center. Further studies are underway to correlate structural changes in 23S rRNA with slower peptide elongation rates observed in mutant strains. These results highlight the important role of L4 and L22 in ribosome structure, function and assembly.