Mutagenesis of Arg335 in bovine mitochondrial elongation factor Tu and the corresponding residue in the Escherichia coli factor affects interactions with mitochondrial aminoacyl-tRNAs.
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.
Hunter
Senyene Eyo
SE
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Spremulli
Linda L
LL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics* / Q000378:metabolism; D001693:Biological Transport, Active / Q000235:genetics; D002417:Cattle; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012335:RNA, Ribosomal / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics* / Q000378:metabolism
D029968:Escherichia coli Proteins; D024101:Mitochondrial Proteins; D012335:RNA, Ribosomal; D012346:RNA, Transfer, Amino Acyl; D001120:Arginine; D010444:Peptide Elongation Factor Tu