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. 2014 Aug 18;15(12):1805-1809.
doi: 10.1002/cbic.201402083. Epub 2014 May 30.

Exploring the substrate range of wild-type aminoacyl-tRNA synthetases

Chenguang Fan #  1 , Joanne M L Ho #  1 , Napon Chirathivat  1 , Dieter Söll  1   2 , Yane-Shih Wang  1
Affiliations

Exploring the substrate range of wild-type aminoacyl-tRNA synthetases

Chenguang Fan et al. Chembiochem. .

Abstract

We tested the substrate range of four wild-type E. coli aminoacyl-tRNA synthetases (AARSs) with a library of nonstandard amino acids (nsAAs). Although these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also show that E. coli tryptophanyl-tRNA synthetase (TrpRS) and tyrosyl-tRNA synthetase have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNA(Trp) altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge in the aim of incorporating multiple different amino acids site-specifically into proteins.

Keywords: aminoacyl-tRNA synthetases; natural nonsense tRNA suppressors; nonstandard amino acids; substrate specificity; synthetic biology; tRNA.

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Figures

Figure 1
Figure 1
Range of substrate specificities of TrpRS. Suppression of the sfGFP-UAG2 and sfGFP-UGA2 genes by the library of nsAA-tRNA was measured by fluorescence intensity. Four wells (A1, A2, I1 and I2) were set as control experiments without adding any nsAA to detect the background signals, which were used to normalize the signals generated by supplied nsAAs. (A) The substrate specificity profile of TrpRS/tRNATrpCCA by production of wild type sfGFP gene. (B) The substrate specificity profile of TrpRS/supU (tRNATrpCUA) by suppression of the sfGFP-UAG2 gene. (C) The substrate specificity profile of TrpRS/trpT (tRNATrpUCA) by suppression of the sfGFP-UGA2 gene. The cell strain JW2580-1 (trpC−) was used in these experiments. The nsAA chemical structures are provided in our reported nsAA library[5b]; Tables S4 and S5 describe the additional nsAAs not previously reported. The labeled nsAA chemical structures are given in Scheme 1.
Figure 2
Figure 2
Range of substrate specificities of TyrRS, LeuRS and SerRS. Suppression of the sfGFP-UAG2 gene by the library of nsAA-tRNACUA was measured by fluorescence intensity. Four wells (A1, A2, I1 and I2) were set as control experiments without adding any nsAA to detect the background signals, which were used to normalize the signals generated by supplied nsAAs. (A) The substrate specificity profile of TyrRS/supF (tRNATyrCUA) pair. (B) The substrate specificity profile of LeuRS/supP (tRNALeuCUA) pair. (C) The substrate specificity profile of SerRS/supD (tRNASerCUA) pair. The nsAA chemical structures are provided in our reported nsAA library[5b]; Tables S4 and S5 describe the additional nsAAs not previously reported. The labeled nsAA chemical structures are given in Scheme 1.
Figure 3
Figure 3
Mass spectrometric analyses of sfGFP proteins. The full length sfGFP-6 was produced by using tRNATrpCUA gene in the Trp auxotrophic stain supplied with 1 mM nsAA 6 in the M9 medium. (A) The LC-MS/MS spectrum of the MXKGEELFTGVVPILVELDGDVNGHK (X denotes nsAA 6 incorporated at Ser2TAG position) fragment from sfGFP-6 protein. (B) The LC-MS/MS spectrum of the ICTTGKLPVPWPTLVTTL fragment from sfGFP-6 protein. b0 and y0 denote loss of water from the b or y fragments; y* denotes loss of ammonia (NH3) from y fragments respectively. The full list of m/z values of fragments is provided in Table S6 and S7.
Scheme 1
Scheme 1
Chemical structures of canonical and non-standard amino acids mentioned in this study.
See this image and copyright information in PMC

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