Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules.
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.
Perla-Kajan
Joanna
J
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, 07101, USA.
Lin
Xin
X
Cooperman
Barry S
BS
Goldman
Emanuel
E
Jakubowski
Hieronim
H
Knudsen
Charlotte R
CR
Mandecki
Wlodek
W
D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006153:Guanosine Diphosphate / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling
D050505:Mutant Proteins; D010455:Peptides; D006153:Guanosine Diphosphate; C012001:polyphenylalanine; D012343:RNA, Transfer; D015951:Factor Xa; D010444:Peptide Elongation Factor Tu