Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe |
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Authors: | Sanderson Lee E Uhlenbeck Olke C |
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Affiliation: | Northwestern University, Department of Biochemistry, Molecular Biology, and Cell Biology, 2205 Tech Drive Hogan 2-100. Evanston, IL, USA. |
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Abstract: | The co-crystal structure of Thermus aquaticus elongation factor Tu.guanosine 5'- [beta,gamma-imido]triphosphate (EF-Tu.GDPNP) bound to yeast Phe-tRNA(Phe) reveals that EF-Tu interacts with the tRNA body primarily through contacts with the phosphodiester backbone. Twenty amino acids in the tRNA binding cleft of Thermus Thermophilus EF-Tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast Phe-tRNA(Phe) determined. Eleven of the 20 mutations reduced the binding affinity from fourfold to >100-fold, while the remaining ten had no effect. The thermodynamically important residues were spread over the entire tRNA binding interface, but were concentrated in the region which contacts the tRNA T-stem. Most of the data could be reconciled by considering the crystal structures of both free EF-Tu.GTP and the ternary complex and allowing for small (1.0 A) movements in the amino acid side-chains. Thus, despite the non-physiological crystallization conditions and crystal lattice interactions, the crystal structures reflect the biochemically relevant interaction in solution. |
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Keywords: | EF-Tu, elongation factor Tu GDPNP, guanosine 5′- [β,γ-imido]triphosphate aa-tRNA, aminoacyl-tRNA TEV, tobacco etch virus PheRS, phenylalanyl-tRNA synthetase |
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