Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

Area of Science:

• Molecular Biology
• Bacterial Ribosome Function
• Protein Synthesis Regulation

Background:

• Trans-translation is a crucial cellular process that rescues ribosomes stalled on aberrant mRNAs.
• Transfer messenger RNA (tmRNA) and small protein B (SmpB) are key components of the trans-translation machinery.
• The precise stoichiometry and location of SmpB during ribosome interaction remain incompletely understood.

Purpose of the Study:

• To elucidate the stoichiometry and ribosomal binding sites of small protein B (SmpB) during trans-translation.
• To investigate the interaction of SmpB with both ribosomal subunits (30S and 50S) and the complete 70S ribosome.
• To propose a mechanistic model for SmpB's role in facilitating tmRNA binding and function.

Main Methods:

• Isolation of SmpB-containing ribosomal complexes.
• Chemical modification of ribosomal RNA using dimethyl sulfate, kethoxal, and hydroxyl radicals.
• Analysis of SmpB footprints on ribosomal RNA to determine binding locations and stoichiometry.

Main Results:

• SmpB binds to the 30S and 50S ribosomal subunits in a 1:1 molar ratio.
• SmpB binds to the 70S ribosome in a 2:1 molar ratio.
• Specific nucleotide footprints indicate SmpB binding near the P-site on the 30S subunit and below the L7/L12 stalk on the 50S subunit.

Conclusions:

• Two molecules of SmpB are proposed to interact with the ribosome during trans-translation.
• The first SmpB molecule likely facilitates tmRNA accommodation by binding to the 50S subunit near the factor-binding site.
• The second SmpB molecule may compensate for the lack of an anticodon stem-loop in tmRNA during later stages of the process.