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

作者：Ieong Ka Weng; Pavlov Michael Y; Kwiatkowski Marek; Ehrenberg Mans^*; Forster Anthony C

来源：RNA-A Publication of the RNA Society, 2014, 20(5): 632-643.

DOI：10.1261/rna.042234.113

• unnatural amino acid
• tRNA(Ala)
• EF-Tu affinity
• ribosome
• translation kinetics

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 similar to 1 mu M EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNAPheB, especially in the case of the bulky bK. In contrast, the swap to the tRNAAlaB 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 EFTu: 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.

• 出版日期2014-5