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. 2007 Oct;13(10):1703-14.
doi: 10.1261/rna.666807. Epub 2007 Aug 13.

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation

Erik A Rodriguez  1 , Henry A Lester, Dennis A Dougherty
Affiliations

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation

Erik A Rodriguez et al. RNA. 2007 Oct.

Abstract

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.

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Figures

FIGURE 1.
FIGURE 1.
Site-specific UAA incorporation. (A) tRNA-dCA-UAA binds EF-1α and/or other components of the translational machinery. (B) UAA is incorporated at the suppression site [XXX(X)], resulting in a protein with an UAA. (C) Undesired hydrolysis and/or aaRS editing results in irreversible loss of the UAA, resulting in tRNA-dCA (76 mer). (D) Injection of the tRNA (lacking dCA, 74 mer) is converted to a 76 mer in vivo by the addition of CA (tRNA-CA). (E) Undesired recognition of the suppressor tRNA-dCA/CA by endogenous aaRS(s) can result in aminoacylation of the tRNA with a natural amino acid. (F) tRNA-dCA/CA-amino acid binds EF-1α and/or other components of the translational machinery. (G) Undesired protein translation can occur by placing a natural amino acid at the suppressor site (XXX[X]), rather than termination at the stop codon or a frameshift, and this competes with UAA incorporation. (H) After translation, the tRNA-dCA/CA is released into the cytoplasm and can repeat steps E–H.
FIGURE 2.
FIGURE 2.
Fits to the Hill equation for β9′UAG+THG73 (74 mer)/-dCA (76 mer) and β9′Q. Filled circles are β9′UAG+THG73 (74 mer) (16 ng per oocyte), open squares are β9′Q, and filled triangles are β9′UAG+THG73-dCA (76 mer) (5 ng per oocyte). EC50 values are 0.24 ± 0.006, 0.31 ± 0.02, and 0.88 ± 0.08 μM ACh, respectively. In each experiment n >3 oocytes.
FIGURE 3.
FIGURE 3.
THG73 mutations and tRNAs studied. Circled positions on THG73 correspond to the knob mutations from Liu et al. (1997), where K1 is C3-G70, K2 is C10-G25, and K3 is G17. The boxed region on THG73 corresponds to the second to fourth positions of the acceptor stem (mutations are shown in gray italics in the right box). Note THG73 K1 and TQAS-3 are the same mutation. Other tRNAs studied are shown at the bottom with only the second to fourth positions of the acceptor stem shown. ENAS and TQAS contain the same nucleotides at these positions.
FIGURE 4.
FIGURE 4.
THG73 knob mutations. Individual tRNA average current were normalized by appropriate THG73 average current and bars represent this average ratio (total of oocytes tested is 91, where each bar is 15>n>5 oocytes). Black bars correspond to tRNA-W (21 ng per oocyte) suppressing at α149UAG and white bars correspond to tRNA (74 mer) (17 ng per oocyte) +β9′UAG in Xenopus Express oocytes. THG73 knob mutations are <20% when suppressing at α149UAG and show they are not functional alternatives for UAA incorporation.
FIGURE 5.
FIGURE 5.
ENAS and ENAS A71 aminoacylation tested at β9′UAG. Individual tRNA (74 mer) (17 ng per oocyte) average current was normalized to THG73 (74 mer) average current and bars represent this average ratio (total of oocytes tested is 75, where each bar is 22>n>5 oocytes). Black and white bars correspond to oocytes from Xenopus Express and Nasco, respectively. ENAS and ENAS A71 show a large amount of aminoacylation in Xenopus Express oocytes after a 2-d incubation, which has not been seen in vitro (Cload et al. 1996) or in vivo (Kleina et al. 1990). Aminoacylation is drastically reduced when tested in Nasco oocytes.
FIGURE 6.
FIGURE 6.
THG73 acceptor stem mutations tested at β9′UAG. Individual tRNA (74 mer) (16 ng per oocyte) average current was normalized to THG73 (74 mer) average current. Bar colors are the same as in Fig. 5. Total of oocytes tested is 189 and each bar is 26>n>8 oocytes. All mutations in the acceptor stem lower aminoacylation in vivo relative to THG73. TQAS-1 shows lack of aminoacylation but is not accepted by the translational machinery (Rodriguez et al. 2007, companion paper, this issue). TQAS′ and TQAS are the most orthogonal tRNAs in Xenopus Express and Nasco oocytes, respectively.
FIGURE 7.
FIGURE 7.
tRNA (74 mer/-dCA) aminoacylation tested at a highly promiscuous site, βA70. All experiments were performed in Nasco oocytes. Black bars are tRNA (74 mer), white bars are tRNA-dCA, and the gray bar is mRNA only. Average currents for TQAS′-dCA and TQAS were normalized to THG73-dCA and THG73, respectively. mRNA only was normalized to THG73. Total of oocytes tested is 60, where each bar is 15>n>6 oocytes with 9 ng of tRNA per oocyte. mRNA only shows significant readthrough of the UAG stop codon. THG73, THG73-dCA, and TQAS′-dCA all show significant aminoacylation when assayed at β70UAG. TQAS shows comparable currents to the injection of mRNA only, showing less aminoacylation of TQAS in Nasco oocytes.
FIGURE 8.
FIGURE 8.
tRNA aminoacylation tested at αK145. Total of oocytes tested is 36, where each dose–response is 17>n>5 oocytes. EC50 values are 167 ± 14, 165 ± 14, 134 ± 15, and 144 ± 2 μM ACh in the order listed in the legend, with 7.5 ng of tRNA per oocyte. All tRNAs are still aminoacylated by Gln and not Lys (wild-type EC50 = 50 μM ACh).
FIGURE 9.
FIGURE 9.
Amber, opal, and frameshift suppressor tRNAs tested at β9′. All tRNA 74 mer or -dCA (7.5 ng per oocyte) average currents were normalized to THG73 74 mer or -dCA average currents, respectively. Black bars are tRNA 74 mer+β9′(UAG, UGA, CGGG, or GGGU) and gray bars are tRNA-dCA (76 mer)+β9′(UAG, UGA, CGGG, or GGGU) in Xenopus Express oocytes. White and hatched bars are tRNA 74 mer and 76 mer, respectively, in Nasco oocytes. Total of tested oocytes is 276, where 17>n>5. TQAS′ and TQAS show significantly reduced aminoacylation in Xenopus Express and Nasco oocytes, respectively, when compared to THG73. Both opal suppressor tRNAs (TQOpS′ and TQOpS) show less aminoacylation than THG73. Overall, the frameshift suppressor YFaFSACCC is the most orthogonal tRNA in both Xenopus Express and Nasco oocytes.
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