PMID: 2508560Nov 1, 1989Paper

Site-directed mutagenesis of the GDP binding domain of bacterial elongation factor Tu

Archives of Biochemistry and Biophysics
Y W HwangD L Miller

Abstract

The tertiary structure model of EF-Tu predicts that the amino acid sequence Val-Asp-His-Gly-Lys-Thr-Thr-Leu (residues 20-27) forms a pocket that binds the pyrophosphate group. To test this model we used site-directed mutagenesis to produce forms of EF-Tu altered in this region. The following mutations were constructed: Gly-20, Val-23, Glu-24, Ile-25, and Pro-27. Each protein was labeled with [35S]Met and was tested for its ability to interact with guanosine nucleotides and EF-Ts. The in vivo activity of each altered protein was tested by determining its ability to confer aurodox sensitivity to a resistant host. Mutations at residues 23, 24, 25, and 27 eliminated the ability of EF-Tu to interact with either guanosine nucleotides or EF-Ts in vitro, and these forms were also inactive in vivo. In contrast, the Gly-20 form was nearly as active as wild-type EF-Tu in vitro and in vivo. This mutation is theoretically equivalent to reversion of the Gly to Val transforming mutation of the cellular form of the ras gene product p21, a protein proposed to be structurally similar to EF-Tu in the GDP binding domain. In contrast to its effect in the ras gene, the Val to Gly conversion did not affect the endogenous GTPase of EF-Tu. We conclude ...Continue Reading

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Citations

Dec 1, 1991·Biochimie·F AbdulkarimD Hughes
Sep 24, 2014·Proceedings of the National Academy of Sciences of the United States of America·Cristina MaracciMarina V Rodnina
Feb 5, 1992·Journal of Molecular Biology·M Kjeldgaard, J Nyborg
Aug 9, 2005·FEBS Letters·Sebastian HanssonDerek T Logan
Jul 18, 1997·The Journal of Biological Chemistry·R N CollinsP Novick
Feb 3, 1998·Molecular and Cellular Biology·C J RichardsonN Segev
Oct 1, 1990·Applied Biochemistry and Biotechnology·J S Dordick

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