Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

Abstract

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an ''orthogonal'' suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA 2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA 2 Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotidedirected mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

“…A corresponding aminoacyl-tRNA synthetase is also required that uniquely recognizes this tRNA and selectively charges it with the unnatural amino acid and no endogenous amino acids. One approach to the generation of orthogonal tRNA-synthetase pairs takes advantage of interspecies differences in tRNA recognition elements (19). For example, Xue and coworkers (20) have shown that B. subtilis tRNA Trp is not a substrate for the tryptophantRNA synthetases from yeast and mammalian cells (21).…”

Expanding tRNA recognition of a tRNA synthetase by a single amino acid change

“…Studies on tRNA identity made clear that the anticodon (position 34-36), the discriminator base (position 73), and base pairs in the acceptor helix are of primary importance in synthetase recognition (19). As these structurally separated nucleotides are recognized by spatially noncontiguous elements in the synthetase protein, a desired change in tRNA binding by this protein may mandate multiple amino acid changes (28,29). The task should be easier in the case of the set of discriminating or nondiscriminating AARSs, because the latter enzyme acylates with its cognate amino acid two different acceptor RNA types.…”

Aminoacyl-tRNA synthetases: Versatile players in the changing theater of translation

“…The structural and mechanistic work summarized above also establishes a critical knowledge base for expansion of the genetic code+ Incorporation of a nonstandard amino acid into proteins in vivo requires both a dedicated suppressor tRNA and a specialized synthetase that is specific for both the new amino acid and tRNA+ 2 Previous experiments showed that misincorporation of amino acid analogs can occur under special conditions+ For example, some mutants of PheRS were isolated that exhibited relaxed specificity for parasubstituted versions of fluoro-phenylalanine (Ibba et al+, 1994), and this has been exploited to explore altered physical-chemical properties of fluorinated proteins (Kirshenbaum et al+, 2002)+ An in vitro approach has also been explored, by which chemically synthesized aminoacyl-tRNA was used to incorporate nonstandard amino acids in b-lactamase (Noren et al+, 1989), ribonuclease A (Jackson et al+, 1994), and nicotinic acid receptor (Nowak et al+, 1995)+ Although these approaches have enjoyed limited success, a robust in vivo system is necessary if extensive characterization of the engineered protein is a key objective+ The first hurdle toward this technology is the development of a mutually compatible aaRS:tRNA pair that is resistant to challenge by competing interactions with the natural amino acid, and noncognate synthetase or tRNA+ Several groups have reported progress in the development of such "orthogonal" aaRS:tRNA pairs that utilize either amber or four base codons for recoding (Liu et al+, 1997;Kowal et al+, 2001)+ The second and more technically demanding step is to create a mutant synthetase capable of efficiently aminoacylating the orthogonal tRNA with a nonstandard amino acid+ Thus far, the only successful strategy reported involves the M. jannaschii TyrRS-tRNA Tyr pair imported into E. coli (Wang et al+, 2001)+ A genetic selection employing combinatorial mutagenesis of selected active site residues in M. jannaschii TyrRS allowed isolation of mutants that suppress an amber-containing reporter gene, when provided with an unnatural amino acid but not tyrosine+ By use of the well-characterized suppression of dihydrofolate reductase, it was demonstrated that the mutant TyRS-tRNA Tyr pair can efficiently insert 29O-methyl tyrosine in response to a stop codon in vivo, although in vitro characterization of the enzyme was limited only to the activation step of the reaction+ Another strategy for incorporating unnatural amino acids into proteins involves the subversion of the editing mechanisms characteristic of selective class I and class II tRNA synthetases+ Using the insertion of cysteine into the thyA gene as a reporter for decreased editing function, mutants of ValRS were selected that contained substitutions in the editing domain (Doring et al+, 2001)+ In addition to mischarging tRNA Val with threonine, a mutant ValRS (T222P) also brought about a significant level of misincorporation of aminobutyrate (up to 24% of valine) when the unnatural amino acid is present at 0+2 mM in the culture media+ This misincorporation by inactivation of the editing site also highlights the less discriminating nature of the synthetic site, and shows how loss of editing can serve as a starting point for selecting synthetases with novel amino acid specificities+ However, there may be nonnatural amino acids for which genetic selections are not possi...…”