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. 2014 May;20(5):632-43.
doi: 10.1261/rna.042234.113. Epub 2014 Mar 26.

A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

Ka-Weng Ieong, Michael Y Pavlov, Marek Kwiatkowski, Måns Ehrenberg, Anthony C Forster

A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

Ka-Weng Ieong et al. RNA. 2014 May.

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.

Keywords: EF-Tu affinity; ribosome; tRNAAla; translation kinetics; unnatural amino acid.

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Figures

FIGURE 1.
FIGURE 1.
A tRNA based on tRNAAla (tRNAAlaB) charged with natural and unnatural AAs. (A) Natural and unnatural L-AAs used in our kinetics studies. (B) Synthetic tRNAAlaB (Zhang et al. 2007), which is an unmodified tRNA based on natural E. coli tRNAAla (black with purple anticodon; tRNA modifications are in green) with changes in blue.
FIGURE 2.
FIGURE 2.
Effects of EF-Tu concentration on the kinetics of dipeptide synthesis from fMet-tRNAifMet and Ala-tRNAAlaB (A,B) or Phe-tRNAAlaB (C,D). Left: Time course (normalized) of f[3H]Met-Ala (A) or f[3H]Met-Phe (C) dipeptide formation at different EF-Tu concentrations in the reaction mixture. Right: (B,D) Hyperbolic fitting of the dependence of the fast phase fraction of the dipeptide formation (A,C) on EF-Tu concentrations. Kd for the binding of Ala-tRNAAlaB and Phe-tRNAAlaB to EF-Tu:GTP were estimated as 0.22 and 0.056 µM, respectively, from the fit. Experiments were done in LS3 buffer at 37°C (see Materials and Methods).
FIGURE 3.
FIGURE 3.
Effects of EF-Tu concentration on the kinetics of dipeptide synthesis from fMet-tRNAifMet and aG-tRNAAlaB (A,B) or mS-tRNAAlaB (C,D).
FIGURE 4.
FIGURE 4.
Effects of EF-Tu concentration on the kinetics of dipeptide synthesis from fMet-tRNAifMet and bK-tRNAAlaB. Experiments were done at 37°C (A,B) and 20°C (C,D).
FIGURE 5.
FIGURE 5.
Effects of EF-Tu concentration on the kinetics of dipeptide synthesis from fMet-tRNAifMet and N-methyl-Phe-tRNAPheB (A) or N-methyl-Phe-tRNAAlaB (B).
FIGURE 6.
FIGURE 6.
Kinetics of the fast phase of GTP hydrolysis and dipeptide synthesis with different AA-tRNA combinations. Time course of GTP hydrolysis (□) and dipeptide synthesis (▪) for dipeptide formation reaction from fMet-tRNAifMet and aG-tRNAAlaB (A), mS-tRNAAlaB (B), Phe-tRNAAlaB (C), Ala-tRNAAlaB (D), or natural Phe-tRNAPhe (E) are shown. Preinitiated ribosomes had Ala codon GCA (A–D) or Phe codon UUC (E) in the A site. All kinetics were measured at 37°C in LS3 buffer. Representative plots are shown for each assay.
FIGURE 7.
FIGURE 7.
Dependence of the rate of fast phase of dipeptide synthesis on ribosome concentration in the reaction mixture. The normalized time course of f[3H]Met-mS (A) or f[3H]Met-bK (B) dipeptide formation for different concentrations of preinitiated ribosomes with the Ala codon (GCA) in the A site (see Materials and Methods). (C) The average times of dipeptide formation τdip, estimated from the experiments in A (□) and B (▪), were plotted versus the inverse of ribosome concentration (Lineweaver-Burke plot); τdipmin- values for dipeptide formation, which is equal to the y-intercept, were estimated from the fits as 13.8 ± 3.7 msec for mS-tRNAAlaB (□) and 13.3 ± 2.3 msec for bK-tRNAAlaB (▪).
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References

    1. Asahara H, Uhlenbeck OC 2002. The tRNA specificity of Thermus thermophilus EF-Tu. Proc Natl Acad Sci 99: 3499–3504 - PMC - PubMed
    1. Bain JD, Glabe CG, Dix TA, Chamberlin AR, Diala ES 1989. Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide. J Am Chem Soc 111: 8013–8014
    1. Bieling P, Beringer M, Adio S, Rodnina MV 2006. Peptide bond formation does not involve acid-base catalysis by ribosomal residues. Nat Struct Mol Biol 13: 423–428 - PubMed
    1. Cload ST, Liu DR, Froland WA, Schultz PG 1996. Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids. Chem Biol 3: 1033–1038 - PubMed
    1. Doi Y, Ohtsuki T, Shimizu Y, Ueda T, Sisido M 2007. Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system. J Am Chem Soc 129: 14458–14462 - PubMed

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