A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein.
A single amino acid substitution (Asp to Asn) at position 138 of Escherichia coli elongation factor Tu (EF-Tu) was introduced in the tufA gene clone by oligonucleotide site-directed mutagenesis. The mutated tufA gene was then expressed in maxicells. The properties of [35S]methionine-labeled mutant and wild type EF-Tu were compared by in vitro assays. The Asn-138 mutation greatly reduced the protein's affinity for GDP; however, this mutation dramatically increased the protein's affinity for xanthosine 5'-diphosphate. The mutant protein forms a stable complex with Phe-tRNA and xanthosine 5'-triphosphate, which binds to ribosomes, whereas it does not form a complex with Phe-tRNA and GTP (10 microM). These results suggest that in EF-Tu.nucleoside diphosphate complexes, amino acid residue 138 must interact with the substituent on C-2 of the purine ring. Thus, in wild type EF-Tu, Asp-138 would hydrogen bond to the 2-amino group of GDP, and in the mutant EF-Tu, Asn-138 would form an equivalent hydrogen bond with the 2-carbonyl group of xanthosine 5'-diphosphate. Aspartic acid 138 is conserved in the homologous sequences of all GTP regulatory proteins. This mutation would allow one to specifically alter the nucleotide specificity of other GTP regulatory proteins.
Hwang
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YW
New York State Office of Mental Retardation and Developmental Disabilities, Staten Island, New York 10314.
Miller
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DL
D001216:Asparagine; D001224:Aspartic Acid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding
D006150:Guanine Nucleotides; D006153:Guanosine Diphosphate; D001224:Aspartic Acid; D001216:Asparagine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu