Fast peptide bond formation and release by the ribosomal large subunit

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Peptide bond formation and peptidyl release occur at the 50S subunit's peptidyl transferase center.
• Existing models of these reactions are based on 50S and 70S ribosome structures.
• Reactions catalyzed by the 50S subunit are significantly slower than those of the 70S ribosome.

Purpose of the Study:

• To investigate methods for accelerating peptide bond formation and peptidyl release reactions catalyzed by the 50S ribosomal subunit.
• To compare the effects of different chemical conditions on these ribosomal functions.
• To provide insights into the evolution of the ribosome and its catalytic mechanisms.

Main Methods:

• Utilized puromycin as a model substrate for peptide bond formation catalyzed by the 50S subunit in the presence of methanol and polyethylene glycol (PEG).
• Investigated peptidyl release reactions using tRNA^Phe or CCA trinucleotide as model substrates on the 50S subunit in acetone.
• Compared reaction rates under various solvent conditions (methanol, PEG, acetone) to assess their impact on substrate binding and catalysis.

Main Results:

• Achieved near-physiological rates of peptide bond formation with puromycin on the 50S subunit in 33% methanol and in aqueous solution with 20% PEG.
• Methanol significantly accelerated substrate binding to the 50S P site, while PEG also enhanced reaction rates.
• Observed fast peptidyl release model reactions in 30% acetone but not with PEG, indicating distinct mechanisms for peptide bond formation and release.
• Reported comparable peptidyl transfer rates between the 50S and 70S subunits under aqueous conditions.

Conclusions:

• Methanol and PEG can significantly enhance the rate of peptide bond formation catalyzed by the 50S ribosomal subunit.
• Different mechanisms govern peptide bond formation and peptidyl release on the 50S subunit.
• The findings support hypotheses about early ribosome evolution and provide a basis for further mechanistic investigations.