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The peptidyl transferase center (PTC) of the ribosome catalyzes peptidyl transfer and release. It consists of domain V of the 23S ribosomal RNA and it is heavily modified by RNA modification enzymes, suggesting these modifications are functionally important. However, individual knockouts (KO) of the enzymes have minimal impacts on bacteria growth, except a two to fourfold deficit for rlmE. To study the significance of the rRNA modifications on cell viability, combinations of KOs are needed. Our collaboratio succeeded in constructing the rluC/rlmE E. coli strain which showed the most severe phenotype yet seen at 37℃ and was lethal at 20℃, suggesting conditional essentiality of the rRNA modification enzymes. Furthermore, an early in vitro reconstitution with 23S rRNA lacking modifications around the PTC “critical region” showed catalytically inert 50S. However, our collaboration constructed a strain with all identified critical region modification enzymes KOed. This strain was viable and displayed a minimal growth deficit at 37℃, suggesting plasticity of the enzymes modifying around the PTC. Although the phenotypes of these KO strains have been well characterized, the molecular explanations for such deficits remain unclear. Here, based on biochemical approaches, I pinpoint that the enzyme KOs affect ribosome assembly and translocation, rather than peptide bond formation or release, in the two combined KO strains. These results clarify the importance and roles of the enigmatic rRNA modifications.

Release is also catalyzed by PTC and understanding the rate-limiting step can help genetic engineering, as readthrough of a stop codon enables the incorporation of unnatural amino acids and treatment of genetic diseases. Although the rate-limiting step was suggested to be hydrolysis at physiological pH, the evidence was indirect. Here, I used fluorine-modified amino acids to activate the ester electrophile. Acceleration of the release reaction with activated ester at lower pHs provides direct evidence for rate-limiting hydrolysis.

Mechanistic studies of peptidyl transfer and release were mainly based on the crystal structures of the 50S subunit. However, both model reactions on the 50S showed orders-of-magnitude slower rates than on the 70S, questioning their relevance. Here, I optimize the peptidyl transfer and release model reactions to near-physiological rates, though in organic solvents. A more physiological solution, achieved by substituting organic solvent with PEG, is found to best accelerate peptidyl transfer, but not release. These optimized reactions should aid analysis of the activities of synthetic ribosomes/PTCs and give insights into the evolution of ribosomes.