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).
Hunter
Senyene Eyo
SE
Department of Chemistry, Lineberger Cancer Research Center, University of North Carolina, Campus Box 3290, Chapel Hill, NC 27599-3290, USA.
Spremulli
Linda L
LL
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012350:RNA, Transfer, Cys / Q000737:chemistry*; D012360:RNA, Transfer, Phe / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D006150:Guanine Nucleotides; D012350:RNA, Transfer, Cys; D012360:RNA, Transfer, Phe; D010444:Peptide Elongation Factor Tu