The Effect of EF-P on tRNA Ribosomal P-site Binding

Abstract

Bacterial elongation factor-P (EF-P) is a conserved protein common to all eubacteria and needed for translation of particular subscripts involved in virulence and antibiotic susceptibility. Similar in size and shape to a tRNA, EF-P undergoes a posttranslatial modification from PoxA that appears to be essential for promoting peptide bond formation. Structurally, EF-P makes contacts near the peptidyl transferase center of the tRNA, as well as with the P-site tRNA. The purpose of this study is to determine whether conserved residues play a role in EF-P’s function. Therefore, EF-P sites that interact with the anticodon stem-loop of the P-site tRNA and the A1339 and G1338 nucleotides in the 16S RNA of the 30S small ribosomal subunit (otherwise known as residues Y183 and R186) were selected for site mutagenesis. When Δefp strains were complemented with the mutants, growth phenotypes could not be rescued, indicating that these residues are critical for function. To verify the mutants were still being aminoacylated by PoxA, in vivo assessment via Western blotting was performed. The results indicate that these mutants are still being aminoacylated, implying that they function at the ribosomal level. Moreover, puromycin assays were performed, the results of which indicate the mutants are not able to properly bind the ribosome.