title: Kinetics of tRNA(Pyl) -mediated amber suppression in Escherichia coli translation reveals unexpected limiting steps and competing reactions.
abstract: The utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNA(Pyl) . Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified Escherichia coli translation system. Dipeptide formation from initiator fMet-tRNA(fMet) and tRNA(Pyl) charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, ∼30-fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF-Tu concentration, allowing measurement of Kd values of EF-Tu binding, both of which were ∼25-fold weaker than normal. However, binding could be increased ∼30-fold by lowering temperature. The fast phase rates were limited by the surprisingly ∼10-fold less efficient binding of EF-Tu:GTP:AA-tRNA(Pyl) ternary complex to the ribosomes, not GTP hydrolysis or peptide bond formation. Furthermore, processivity was unexpectedly impaired as ∼40% of the dipeptidyl-tRNA(Pyl) could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non-cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. Biotechnol. Bioeng. 2016;113: 1552-1559. © 2015 Wiley Periodicals, Inc.
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. a.forster@icm.uu.se.
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Forster
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics*; D016153:Genes, Suppressor; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism
chemical_list: D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet); D010444:Peptide Elongation Factor Tu
keywords: EF-Tu; amber suppression; kinetics; protein synthesis; release factor 2; tRNAPyl