title: A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids.
abstract: There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At ∼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.
authors.forename: Ka-Weng
authors.initials: KW
authors.lastname: Ieong
authors.forename: Michael Y
authors.initials: MY
authors.lastname: Pavlov
authors.forename: Marek
authors.initials: M
authors.lastname: Kwiatkowski
authors.forename: Måns
authors.initials: M
authors.lastname: Ehrenberg
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Forster
mesh_terms: D000596:Amino Acids / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000235:genetics; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*
chemical_list: D000596:Amino Acids; D012346:RNA, Transfer, Amino Acyl; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu
keywords: EF-Tu affinity; ribosome; tRNAAla; translation kinetics; unnatural amino acid