Toward a model for the interaction between elongation factor Tu and the ribosome

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Bacterial protein synthesis relies on elongation factors and nucleotide hydrolysis.
• Elongation factor-Tu (EF-Tu) plays a crucial role in delivering aminoacyl-tRNA (aa-tRNA) to the ribosome.
• GTP hydrolysis by EF-Tu is essential for aa-tRNA selection and polypeptide elongation.

Purpose of the Study:

• To investigate the stoichiometry of nucleotide hydrolysis during bacterial protein synthesis.
• To understand the role of EF-Tu in GTP hydrolysis and aa-tRNA binding.
• To characterize the impact of EF-Tu mutations on substrate specificity and reaction stoichiometry.

Main Methods:

• Site-directed mutagenesis of EF-Tu (Asp138 to Asn).
• Assays measuring nucleotide hydrolysis (GTP and xanthosine triphosphate).
• Analysis of aminoacyl-tRNA binding to mRNA-programmed ribosomes.

Main Results:

• Substitution of Asp138 with Asn in EF-Tu altered substrate specificity from GTP to xanthosine triphosphate.
• The EF-Tu-mediated reactions were shown to involve the hydrolysis of two nucleotide triphosphates per incorporated phenylalanine.
• This stoichiometry of two is linked to the accurate binding of the cognate aa-tRNA to the ribosome.

Conclusions:

• The study elucidates the precise stoichiometry of nucleotide hydrolysis in EF-Tu-mediated aa-tRNA binding.
• EF-Tu's role in protein synthesis involves a two-nucleotide hydrolysis event for correct aa-tRNA selection.
• Understanding this mechanism provides insights into the fidelity of bacterial translation.