Conserved Discrimination against Misacylated tRNAs by Two Mesophilic Elongation Factor Tu Orthologs

Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNAGln and Asn-tRNAAsn by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNAGln and Asp-tRNAAsn as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNAAsn and predicted a similar discriminatory response against Glu-tRNAGln [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194−6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420−3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNAGln or Asp-tRNAAsn, and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNAGln, like Asp-tRNAAsn, will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.