Interrupted catalysis: the EF4 (LepA) effect on back-translocation.

Area of Science:

• Bacterial protein biosynthesis
• Ribosome function and dynamics
• Molecular mechanisms of translation

Background:

• Elongation factor G (EF-G) facilitates ribosome translocation during protein synthesis.
• Elongation factor 4 (EF4) shares structural similarity with EF-G but mediates back-translocation.
• EF4 plays a role in bacterial growth under specific conditions.

Purpose of the Study:

• To elucidate the kinetic mechanism of EF4-catalyzed back-translocation.
• To investigate the impact of translocation inhibitors (spectinomycin, viomycin) on EF4 activity.
• To understand the role of EF4 in optimizing protein biosynthesis.

Main Methods:

• In vitro kinetic assays measuring puromycin reactivity of peptidyl-tRNA.
• Fluorescence-based monitoring of labeled tRNAs and mRNA.
• Analysis of EF4-dependent transitions between ribosomal complex states (POST, PRE).

Main Results:

• EF4-mediated back-translocation follows a four-step kinetic pathway (POST → I(1) → I(2) → I(3) → PRE).
• tRNA and mRNA movements are coupled, but sometimes decoupled from the peptidyl-tRNA 3'-end.
• EF4 catalysis appears interrupted, pausing at an intermediate state (I(3)) rather than completing back-translocation.

Conclusions:

• EF4-catalyzed back-translocation is a multi-step process distinct from the reverse of EF-G-mediated translocation.
• The intermediate state I(3) accumulation likely regulates polypeptide elongation.
• EF4's interrupted catalysis contributes to efficient and accurate protein synthesis.