An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

Area of Science:

• Bacterial protein synthesis
• Molecular mechanisms of translation
• Ribosome rescue pathways

Background:

• Bacteria utilize transfer-messenger RNA (tmRNA) and its partner protein SmpB to rescue ribosomes stalled on truncated messenger RNAs.
• The tmRNA-SmpB complex mimics tRNA and is delivered to the ribosome by elongation factor Tu (EF-Tu).
• SmpB binding to the ribosomal decoding center is essential for tmRNA entry, but the mechanism of EF-Tu activation without canonical codon-anticodon interaction remains unclear.

Purpose of the Study:

• To investigate the role of the interaction between SmpB residue His136 and 16S rRNA nucleotide G530 in bacterial ribosome rescue.
• To elucidate the mechanism of EF-Tu activation and GTP hydrolysis during tmRNA-mediated ribosome rescue.

Main Methods:

• Utilized pre-steady-state kinetic methods to study the interaction.
• Employed site-directed mutagenesis of SmpB (His136 mutation) to assess its impact on ribosome rescue.
• Measured GTP hydrolysis rates and peptide bond formation kinetics.

Main Results:

• Mutation of SmpB His136 did not affect SmpB's affinity for the ribosomal A-site.
• The His136 mutation significantly reduced the rate of GTP hydrolysis by EF-Tu.
• Peptide bond formation rate was minimally affected by the His136 mutation, suggesting EF-Tu release precedes GTP hydrolysis.

Conclusions:

• The interaction between SmpB His136 and 16S rRNA G530 is critical for EF-Tu GTP hydrolysis during tmRNA-mediated ribosome rescue.
• EF-Tu likely dissociates from the tmRNA-SmpB complex before GTP hydrolysis.
• These findings provide insights into the initial steps of the ribosome rescue pathway.