Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Elongation factor Tu (EF-Tu) plays a crucial role in bacterial protein synthesis by delivering aminoacyl-tRNAs to the ribosome.
• Understanding the precise binding interactions of EF-Tu with ribosomal subunits is key to elucidating the mechanism of translation.

Purpose of the Study:

• To investigate the formation and characteristics of a complex between EF-Tu, GTP, aminoacyl-tRNA, and the 30S ribosomal subunit.
• To determine the binding site and requirements for EF-Tu complex formation on the 30S subunit.
• To analyze the subsequent reaction of this complex with the 50S ribosomal subunit.

Main Methods:

• Formation and isolation of a ternary complex of EF-Tu, GTP, and phenylalanyl-tRNA (Phe-tRNA) with oligo(uridylic acid) [oligo(U)] and the 30S ribosomal subunit of Escherichia coli.
• Biochemical assays to characterize the binding site and stoichiometry of EF-Tu on the 30S subunit.
• Investigating the effect of adding 50S ribosomal subunits to the preformed 30S-EF-Tu complex, monitoring GTP hydrolysis and EF-Tu release.

Main Results:

• A stable complex of EF-Tu, GTP, Phe-tRNA, oligo(U), and the 30S ribosomal subunit was formed and isolated, binding to the functionally active 30S site.
• EF-Tu binding to the 30S subunit required both aminoacyl-tRNA and cognate messenger RNA (oligo(U)).
• Addition of 50S subunits to the 30S-EF-Tu complex induced rapid GTP hydrolysis and stoichiometric release of EF-Tu, forming 70S ribosomes.

Conclusions:

• EF-Tu ternary complex binds to isolated 30S subunits at the same site occupied during interaction with the 70S ribosome.
• The reaction of the 30S-EF-Tu complex with 50S subunits mimics the final steps of elongation on the 70S ribosome.
• This binding to isolated 30S subunits does not occur during the elongation microcycle, suggesting a specific context-dependent role.