Characterizing L4 and L22 mutations in Escherichia coli strains resistant to Oleandomycin Triacetate

Abstract

Ribosomes, essential to the life of the cell, are the site of protein synthesis. The bacterial ribosome contains two dissimilar subunits, 30S and 50S, that consist of rRNA molecules and proteins (ribosomal proteins). During translation, the ribosome mediates precise binding of the mRNA codon to the anticodon of a tRNA molecule containing a specific amino acid, and then catalyzes addition of the amino acid to the growing peptide chain. Macrolide antibiotics, such as erythromycin, inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit in the peptide exit channel. Interestingly, many ribosomal proteins such as L4 and L22 have extensions (tentacles) that reach into the mass of rRNA. The ends of the tentacles of L4 and L22 form a constriction in the peptide exit tunnel. Mutations in the L4 and L22 tentacles result in erythromycin resistance in E.coli, either because the antibiotic cannot bind (L4 mutant) or because it binds but cannot inhibit protein synthesis (L22 mutant). To learn more about the role of the tentacles in ribosome function and synthesis, we are isolating mutants of E. coli that are resistant to oleandomycin triacetate, a macrolide related to erythromycin. Mutants shown to have a change in the L4 or L22 gene will be characterized further. For example, assays will determine temperature sensitivity, growth rate, and rate of peptide elongation. The effect of the mutations on ribosome assembly and accuracy of translation will also be analyzed. (This research is supported by NSF Grant MCB-0349443 and MARC U*STAR Grant GM 08663 from NIH.)