PortEco logo

PMID:20083494

From EcoliWiki
Jump to: navigation, search

Contents

Citation

Perla-Kajan, J, Lin, X, Cooperman, BS, Goldman, E, Jakubowski, H, Knudsen, CR and Mandecki, W (2010) Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules. Protein Eng. Des. Sel. 23:129-36

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.

Links

PubMed PMC2816605 Online version:10.1093/protein/gzp079

Keywords

Binding Sites; Electrophoretic Mobility Shift Assay; Escherichia coli/enzymology; Escherichia coli/genetics; Factor Xa/metabolism; Fluorescence Resonance Energy Transfer; Guanosine Diphosphate/metabolism; Hydrolysis; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins/chemistry; Mutant Proteins/genetics; Mutant Proteins/isolation & purification; Mutant Proteins/metabolism; Mutation; Nucleic Acid Conformation; Peptide Elongation Factor Tu/chemistry; Peptide Elongation Factor Tu/genetics; Peptide Elongation Factor Tu/isolation & purification; Peptide Elongation Factor Tu/metabolism; Peptides/metabolism; Protein Conformation; Protein Engineering; RNA, Transfer/chemistry; RNA, Transfer/metabolism; Sequence Analysis, DNA; Staining and Labeling

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

See also

References

See Help:References for how to manage references in EcoliWiki.