Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements
2015 (English)In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 10, no 10, 2187-2192 p.Article in journal, Letter (Refereed) Published
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.
Place, publisher, year, edition, pages
2015. Vol. 10, no 10, 2187-2192 p.
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-262591DOI: 10.1021/acschembio.5b00335ISI: 000363225100002PubMedID: 26191973OAI: oai:DiVA.org:uu-262591DiVA: diva2:854502
FunderSwedish Research CouncilSwedish Research Council