Construction of a fully active Cys-less elongation factor Tu: functional role of conserved cysteine 81

Biochim Biophys Acta. 2011 May;1814(5):684-92. doi: 10.1016/j.bbapap.2011.02.007. Epub 2011 Feb 19.


In order to study the structural and functional requirements of the essential translational GTPase elongation factor (EF) Tu for efficient and accurate ribosome-dependent protein synthesis, construction of a cysteine-free (Cys-less) mutant variant allowing for the site-directed introduction of fluorescent and non-fluorescent labels is of great importance. However, previous reports suggest that a cysteine residue in position 81 of EF-Tu from Escherichia coli is essential for its function. To study the functional role of cysteine 81 and to construct a fully active Cys-less EF-Tu, we have analyzed 125 bacterial sequences with respect to sequence variations in this position revealing that in a small number of sequences alanine and methionine can be found. Here we report the detailed comparative biochemical analysis of three Cys-less variants of EF-Tu containing these substitutions as well as the isosteric amino acid serine. By characterizing nucleotide binding, EF-Ts interaction, aminoacyl-tRNA binding, and delivery to the ribosome, we demonstrate that only alanine (or cysteine) can be tolerated in this position and that the serine and methionine substitutions significantly impair aminoacyl-tRNA, but not nucleotide binding. Our findings suggest a critical functional role of the amino acid residue in position 81 of EF-Tu with respect to aminoacyl-tRNA binding. Based on structural considerations, we suggest that position 81 indirectly contributes to aminoacyl-tRNA binding through the accurate positioning of helix B.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cysteine / chemistry*
  • Cysteine / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Elongation Factor Tu / chemistry*
  • Peptide Elongation Factor Tu / genetics
  • Peptide Elongation Factor Tu / metabolism*
  • Protein Structure, Secondary
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship


  • Peptide Elongation Factor Tu
  • Cysteine