title: Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements.
abstract: 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.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University , Husargatan 3, Box 596, Uppsala 75124, Sweden.
authors.forename: Jinfan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University , Husargatan 3, Box 596, Uppsala 75124, Sweden.
authors.forename: Marek
authors.initials: M
authors.lastname: Kwiatkowski
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University , Husargatan 3, Box 596, Uppsala 75124, Sweden.
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Forster
mesh_terms: D002384:Catalysis; D007700:Kinetics; D008954:Models, Biological; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D058105:Polymerization; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D010455:Peptides; D012346:RNA, Transfer, Amino Acyl