Structure-function relationships of elongation factor Tu as studied by mutagenesis

Area of Science:

• Molecular Biology
• Protein Biochemistry
• Structural Biology

Background:

• Elongation factor Tu (EF-Tu) is crucial for protein synthesis, binding GTP/GDP and catalyzing GTP hydrolysis.
• The N-terminal G domain of EF-Tu is key for nucleotide binding and GTPase activity, free from allosteric constraints.
• Mutagenesis studies focus on the GTP-binding pocket to elucidate EF-Tu's function-structure relationships.

Purpose of the Study:

• To investigate structure-function relationships of elongation factor Tu (EF-Tu) through targeted mutagenesis.
• To analyze the role of specific residues within the G domain and GTP-binding pocket in EF-Tu activity.
• To assess the impact of mutations on GTP binding, GTP hydrolysis, and protein synthesis fidelity.

Main Methods:

• Site-directed mutagenesis of the Escherichia coli tufA gene encoding EF-Tu.
• Isolation and characterization of the N-terminal G domain of EF-Tu.
• Assays to measure GTP/GDP binding, GTP hydrolysis rates, and protein synthesis activity (poly(Phe) synthesis).

Main Results:

• Mutations VG20, PT82, HG84, and SD173 significantly affected GTP hydrolysis, GDP/GTP exchange, or nucleotide binding.
• Residues Gln114 and Glu117 influenced GTPase activity and G domain stability, respectively.
• The double mutation VD88/LK121 reduced G domain stability without majorly impacting GTPase activity.
• EF-TuVG20 exhibited reduced poly(Phe) synthesis but enhanced accuracy, likely due to ribosomal pausing.

Conclusions:

• Specific residues in EF-Tu's G domain are critical for regulating GTPase activity, nucleotide binding, and protein synthesis fidelity.
• Mutations can decouple GTPase activity from nucleotide binding or alter the balance between synthesis efficiency and accuracy.
• Understanding these structure-function relationships provides insights into the molecular mechanisms of translation.