Effects of mutagenesis of Gln97 in the switch II region of Escherichia coli elongation factor Tu on its interaction with guanine nucleotides, elongation factor Ts, and aminoacyl-tRNA.
Elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA to the A-site of the ribosome. In a multiple-sequence alignment of prokaryotic EF-Tu's, Gln97 is nearly 100% conserved. In contrast, in mammalian mitochondrial EF-Tu's, the corresponding position is occupied by a conserved proline residue. Gln97 is located in the switch II region in the GDP/GTP binding domain of EF-Tu. This domain undergoes a significant structural rearrangement upon GDP/GTP exchange. To investigate the role of Gln97 in bacterial EF-Tu, the E. coli EF-Tu variant Q97P was prepared. The Q97P variant displayed no activity in the incorporation of [(14)C]Phe on poly(U)-programmed E. coli ribosomes. The Q97P variant bound GDP more tightly than the wild-type EF-Tu with K(d) values of 7.5 and 12 nM, respectively. The intrinsic rate of GDP exchange was 2-3-fold lower for the Q97P variant than for wild-type EF-Tu in the absence of elongation factor Ts (EF-Ts). Addition of EF-Ts equalized the GDP exchange rate between the variant and wild-type EF-Tu. The variant bound GTP at 3-fold lower levels than the wild-type EF-Tu. Strikingly, the Q97P variant was completely inactive in ternary complex formation, accounting for its inability to function in polymerization. The structural basis of these observations is discussed.
Navratil
Tomas
T
Department of Chemistry, Campus Box 3290, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Spremulli
Linda L
LL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; C023512:elongation factor Ts; D005973:Glutamine; D006153:Guanosine Diphosphate; D011072:Poly U; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu