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Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules.


ABSTRACT: Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

SUBMITTER: Perla-Kajan J 

PROVIDER: S-EPMC2816605 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules.

Perla-Kajan Joanna J   Lin Xin X   Cooperman Barry S BS   Goldman Emanuel E   Jakubowski Hieronim H   Knudsen Charlotte R CR   Mandecki Wlodek W  

Protein engineering, design & selection : PEDS 20100118 3


Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions  ...[more]

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