Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements

• Published in: ACS chemical biology Vol. 10; no. 10; pp. 2187 - 2192
• Main Authors: Wang, Jinfan, Kwiatkowski, Marek, Forster, Anthony C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.10.2015
• Subjects: Biochemistry; Biokemi; Catalysis; Kinetics; Models, Biological; Peptides - chemical synthesis; Peptides - chemistry; Peptides - metabolism; Polymerization; Ribosomes - metabolism; RNA, Transfer, Amino Acyl - chemistry
• ISSN: 1554-8929; 1554-8937; 1554-8937
• DOI: 10.1021/acschembio.5b00335

Ribosomal synthesis of polymers of unnatural amino acids (AAs) is limited by low incorporation efficiencies using the artificial AA-tRNAs, but the kinetics have yet to be studied. Here, kinetics were performed on five consecutive incorporations using various artificial AA-tRNAs with all intermediate products being analyzed. Yields within a few seconds displayed similar trends to our prior yields after 30 min without preincubation, demonstrating the relevance of fast kinetics to traditional long-incubation translations. Interestingly, the two anticodon swaps were much less inhibitory in the present optimized system, which should allow more flexibility in the engineering of artificial AA-tRNAs. The biggest kinetic defect was caused by the penultimate dC introduced from the standard, chemoenzymatic, charging method. This prompted peptidyl-tRNA drop-off, decreasing processivities during five consecutive AA incorporations. Indeed, two tRNA charging methods that circumvented the dC dramatically improved efficiencies of ribosomal, consecutive, unnatural AA incorporations to give near wild-type kinetics.