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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

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

Biochemistry 42(46): 13587-13595

ISSN/ISBN: 0006-2960

PMID: 14622005

DOI: 10.1021/bi034855a

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.

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Accession: 009830327

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