Mutagenesis of glutamine 290 in Escherichia coli and mitochondrial elongation factor Tu affects interactions with mitochondrial aminoacyl-tRNAs and GTPase activity.

Elongation factor Tu (EF-Tu) is responsible for the delivery of the aminoacyl-tRNAs (aa-tRNA) to the ribosome during protein synthesis. The primary sequence of domain II of EF-Tu is highly conserved. However, several residues thought to be important for aa-tRNA binding in this domain are not conserved between the mammalian mitochondrial and bacterial factors. One of these residues is located at position 290 (Escherichia coli numbering). Residue 290 is Gln in most of the prokaryotic factors but is conserved as Leu (L338) in the mammalian mitochondrial factors. This residue is in a loop contacting the switch II region of domain I in the GTP-bound structure. It also helps to form the binding pocket for the 5' end of the aa-tRNA in the ternary complex. In the present work, Leu338 was mutated to Gln (L338Q) in EF-Tu(mt). The complementary mutation was created at the equivalent position in E. coli EF-Tu (Q290L). EF-Tu(mt) L338Q functions as effectively as wild-type EF-Tu(mt) in poly(U)-directed polymerization with both prokaryotic and mitochondrial substrates and in ternary complex formation assays with E. coli aa-tRNA. However, the L338Q mitochondrial variant has a reduced affinity for mitochondrial Phe-tRNA(Phe). E. coli EF-Tu Q290L is more active in poly(U)-directed polymerization with both mitochondrial and prokaryotic substrates and has a higher GTPase activity in both the absence and presence of ribosomes. Surprisingly, while E. coli EF-Tu Q290L is more active in polymerization with mitochondrial Phe-tRNA(Phe), this variant has low activity in the formation of a stable ternary complex with mitochondrial aa-tRNA.

Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu.

During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA(Phe). In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe).

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.

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNAs.

Elongation factor Tu (EF-Tu) binds GTP and aminoacyl-tRNA (aa-tRNA) forming a ternary complex which is delivered to the A-site of the ribosome. Animal mitochondrial tRNAs are quite unusual and lack many of the residues important for the stabilization of the structures of other tRNAs. The stabilities of the ternary complexes formed by mammalian mitochondrial and E. coli EF-Tu were determined with four bovine mitochondrial aa-tRNAs. The ternary complex with Phe-tRNA(Phe) has a Kd of about 75 nM. Equilibrium dissociation constants are tightest for the two native Ser-tRNA species (17 nM). Ternary complexes formed with the transcript of tRNA(AGY)Ser are 10-fold weaker than those formed with the native tRNA.