We tested the substrate range of four wild-type aminoacyl-tRNA synthetases (AARSs)

We tested the substrate range of four wild-type aminoacyl-tRNA synthetases (AARSs) with a library of nonstandard amino acids (nsAAs). sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge to the goal of incorporating multiple different amino acids site-specifically into proteins. experiments with a mixture of all AARSs showed that more Tirapazamine than 90 nsAAs can be acylated onto Tirapazamine tRNA[3]. Protein engineering directed toward expansion of the genetic code during the past decade[4] led to many variant AARS enzymes with substrate polyspecificity for some of the nsAAs employed[4a 5 Some variants of tyrosyl-tRNA synthetase (TyrRS)[5i] and of pyrrolysyl-tRNA synthetase can acylate more than ten different compounds[5e 5 Therefore the high specificity observed by the AARSs against the other canonical amino acids Tirapazamine does not extend to a large number of nsAAs. We decided to examine a number of canonical wild-type AARSs for their substrate specificity with a large library of nsAAs. As test enzymes we used leucyl-tRNA synthetase (LeuRS) seryl-tRNA synthetase (SerRS) tryptophanyl-tRNA synthetase (TrpRS) and TyrRS. Each of these enzymes has a natural nonsense suppressor tRNA[6] that when acylated can serve to insert Tirapazamine its amino acid in response to a stop codon (auxotrophic strains for the cognate amino acid[7]. Our results lend further support to our view of natural AARSs being polyspecific for nsAAs. Results Experimental scheme We used the wild-type AARS enzymes with their native amber or opal suppressor tRNAs[6]. They were Mouse monoclonal to APOA4 (tRNATrpCUA) (tRNATrpTyrUCA) (tRNA) (tRNALeuCUA CUA) and Tirapazamine (tRNASerCUA). The TyrRS/pair was used to optimize the fluorescence signal arising from read-through of the mRNA in a auxotrophic stain (Figure S1). The auxotrophic strains (Table S1) transformed with the genes for the AARS/suppressor tRNA pair (Figure S2 and Table S2) were grown at 37°C for 16 h in minimal medium with 1% glycerol supplemented with the 20 canonical amino acids (1 mM). After washing to remove the amino acids and transfer to minimal medium the strains were cultured in a 384-well plate format where each well contained one member of our nsAA library at 1 mM concentration. The nsAAs library contained 313 members of which 272 were previously reported [5b] (Tables S3-S5). The fluorescence readout which was contingent on suppression of the mRNA was used as a measure of the ability of the corresponding endogenous AARS (LeuRS SerRS TrpRS and TyrRS) to recognize the nsAA substrates. This allowed a measurement of the range of amino acid substrate specificity for the AARS. The wild-type protein sequence of sfGFP contains a single Trp (1) residue (W57). Under our incorporation conditions with wild-type tRNATrpCCA this W57 could also be replaced by Trp analogs (2-4) Cys analog 5 and Phe analog 6 (Scheme 1; Figure 1A). The fluorescence intensities generated by nsAAs 4 and 5 were comparable to that of Trp. A similar substrate preference was observed with the TrpRS/pair (Figure 1B) although the pattern of suppression efficiencies was different. This change in substrate range stemmed purely from the different anticodon sequence since the tRNA differs only by a single base change from the endogenous tRNATrpCCA isoacceptor.[6] We Tirapazamine also tested the TrpRS substrate range with the natural opal suppressor tRNATrpUCA encoded by pair incorporated Trp (1) into sfGFP but at a lower efficiency (Figure 1C). Interestingly a Trp lactic acid analog (2) (Figure 1C) was a better substrate than Trp for TrpRS/pair. Thus different tRNAs gave somewhat different suppression profiles suggesting that the nature of the anticodon may affect the amino acid recognition a property that was also seen with glutaminyl-tRNA synthetase[8]. This suggests that in the strategy to develop orthogonal translation systems for genetic code expansion modulations in amino acid recognition should be considered when tRNA anticodons are altered. Figure 1 Range of substrate specificities of TrpRS. Suppression of the and 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 … Scheme 1 Chemical structures of canonical and non-standard amino acids mentioned in this study. The substrate specificity profile of TyrRS/pair indicated that eight aromatic ring-containing nsAAs (3-4 7 were substrates with appreciable signals but had low suppression.