Mutagenesis of Arg335 in bovine mitochondrial elongation factor Tu and the corresponding residue in the Escherichia coli factor affects interactions with mitochondrial aminoacyl-tRNAs

RNA Biol. 2004 Jul;1(2):95-102. doi: 10.4161/rna.1.2.1034. Epub 2004 Jul 16.


During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of the ribosome. Mammalian mitochondrial EF-Tu (EF-Tu(mt)) carries out this activity using aa-tRNAs that lack many of the invariant or semi-invariant residues that stabilize the 3-dimensional structures of canonical tRNAs. The primary sequence of EF-Tu is highly conserved. However, several residues involved in aa-tRNA binding are not conserved between the mitochondrial and bacterial factors. One such residue, located at position 287 in Escherichia coli EF-Tu, is adjacent to the 5' end of the aa-tRNA and is acidic in all prokaryotic factors but is basic in EF-Tu(mt). Site-directed mutagenesis of this residue (Glu287) in E. coli EF-Tu and complementary mutagenesis of the corresponding Arg335 in EF-Tu(mt) was performed to create E. coli EF-Tu E287R and EF-Tu(mt) R335E respectively. EF-Tu(mt) R335E has a reduced activity in ternary complex formation and A-site binding with mitochondrial Phe-tRNA.(Phe) In contrast, E. coli EF-Tu E287R is more active that the wild-type factor in forming ternary complexes with mitochondrial Phe-tRNA,(Phe) and the variant promotes the binding of mitochondrial aa-tRNA to the ribosome more effectively than does the wild-type factor. Both EF-Tu(mt) R335E and E. coli EF-Tu E287R have activities comparable to the corresponding wild-type factors in assays using E. coli Phe-tRNA.(Phe) These data suggest that the residue at position 287 plays an important role in the binding and EF-Tu-mediated delivery of mitochondrial aa-tRNAs to the A-site of the ribosome.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Arginine / genetics*
  • Arginine / metabolism
  • Biological Transport, Active / genetics
  • Cattle
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Mitochondrial Proteins / genetics*
  • Mitochondrial Proteins / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Peptide Elongation Factor Tu / genetics*
  • Peptide Elongation Factor Tu / metabolism
  • Protein Binding / genetics
  • RNA, Ribosomal / metabolism
  • RNA, Transfer, Amino Acyl / genetics*
  • RNA, Transfer, Amino Acyl / metabolism


  • Escherichia coli Proteins
  • Mitochondrial Proteins
  • RNA, Ribosomal
  • RNA, Transfer, Amino Acyl
  • Arginine
  • Peptide Elongation Factor Tu