Analyses of assembly and functional defects in the ribosome of L4 and L22 mutant Escherichia coli strains

The ribosome is a complex macromolecule consisting of RNA and protein subunits that is responsible for translating the genetic code and protein synthesis. Within its large subunit, the exit tunnel exists as a conduit for nascent peptides to traverse before reaching the cytoplasm or membrane translocon. The tips of the extended loops (also called tentacles) of two proteins, L4 and L22, contribute to the surface of the narrowest portion of Escherichia coli's exit tunnel. Mutations in the tentacles of the L4 and L22 proteins promote resistance to a class of antibiotics referred to as macrolides. Although the mutant strains have the advantage of growing in the presence of the antibiotic, erythromycin, they have the disadvantage of growing slower than the wildtype. The decreased rate of growth may be a reflection of structural changes within the 23S rRNA component of the large subunit induced by structural changes in L4 and L22, which in turn result in defects in ribosomal assembly and/or peptide synthesis.;My research involved determining whether the reduced growth rate can be attributed to the rate of peptide bond formation in the peptidyltransferase (PT) center and/or the reduced efficiency of ribosomal assembly. I used the peptidyltransferase assay on total ribosomes and purified subunits to evaluate the effect of the L4 and L22 mutations on ribosomal function. A decrease in PT activity was observed amongst all mutants. Analyses of Variance and Tukey's honestly significant difference test indicated that the differences between the wildtype and the mutants were statistically significant. Additional statistical analyses revealed a strong positive correlation between my PT results and Zaman's data in which she used the beta-galactosidase induction assay to show a decreased rate of peptide chain elongation amongst the mutants.;I also used sucrose gradient analyses and primer extension to evaluate the effect of L4 and L22 mutations on ribosomal assembly and processing. An abnormal peak was observed between the 30S and 50S peaks in the selected mutants. Increased levels of precursor 23S rRNA were observed in the primer extension results of all mutants. My results further substantiate the important roles that the L4 and L22 proteins provide in the function and assembly of the ribosome.