Fast peptide bond formation and release by the ribosomal large subunit

Peptide bond formation and peptidyl release are catalyzed at the peptidyl transferase center of the 50S subunit of the 70S ribosome. Proposed catalytic mechanisms at the peptidyl transferase center are based on structures of model substrates bound to the 50S and the 70S. Yet, peptidyl transfer and release reactions catalyzed by the 50S are slower by >3 orders of magnitude than those of the 70S. Here, we obtained a near-physiological rate of peptide bond formation with puromycin catalyzed by the 50S in 33% methanol at 37 °C, and fast rates were even attained in aqueous solution using 20% PEG. Interestingly, methanol, not PEG, accelerated the reaction by stimulating substrate binding just to the 50S P site. In addition, we obtained fast peptidyl release model reactions catalyzed by tRNA^Phe or Cytosine-Cytosine-Adenine (CCA) trinucleotide on the 50S in 30% acetone. However, PEG did not enable the release reaction, suggesting different mechanisms for release and peptide bond formation. The now-comparable peptidyl transfer rates of the 50S and 70S under aqueous conditions strengthen mechanistic proposals, give credence to hypothetical progenitor ribosomes before evolution of the 30S and will aid mechanistic investigations with model substrates or ancestral subsets of the ribosome.