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A General Approach for the Generation of Orthogonal Trnas

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A General Approach for the Generation of Orthogonal Trnas Chemistry & Biology 8 (2001) 883^890 www.elsevier.com/locate/chembiol Research Paper A general approach for the generation of orthogonal tRNAs Lei Wang a; 1, Peter G. Schultz b; * aDepartment of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA bDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Received 26 March 2001; accepted 29 May 2001 First published online 27 July 2001 Abstract Background: The addition of new amino acids to the genetic cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four code of Escherichia coli requires an orthogonal suppressor tRNA mutant suppressor tRNAs were selected that are poorer substrates Tyr that is uniquely acylated with a desired unnatural amino acid by for E. coli synthetases than M. jannaschii tRNACUA, but still can Tyr an orthogonal aminoacyl-tRNA synthetase. A tRNACUA^tyrosyl- be charged efficiently by M. jannaschii TyrRS. Tyr tRNA synthetase pair imported from Methanococcus jannaschii Conclusions: The mutant suppressor tRNACUA together with can be used to generate such a pair. In vivo selections have been the M. jannaschii TyrRS is an excellent orthogonal tRNA^ developed for selecting mutant suppressor tRNAs with enhanced synthetase pair for the in vivo incorporation of unnatural amino orthogonality, which can be used to site-specifically incorporate acids into proteins. This general approach may be expanded to unnatural amino acids into proteins in E. coli. generate additional orthogonal tRNA^synthetase pairs as well as Results: A library of amber suppressor tRNAs derived from M. probe the interactions between tRNAs and their cognate Tyr Tyr jannaschii tRNA was generated. tRNACUAs that are substrates synthetases. ß 2001 Elsevier Science Ltd. All rights reserved. for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of Keywords: Aminoacyl-tRNA synthetase; In vivo selection; In amber nonsense mutations in the barnase gene. The remaining vivo unnatural amino acid mutagenesis; Orthogonal suppressor Tyr tRNACUAs were then selected for their ability to suppress amber tRNA nonsense codons in the L-lactamase gene in the presence of the 1. Introduction tRNAs, but e¤ciently charges the orthogonal suppressor tRNA. The speci¢city of this synthetase is then altered so In vitro methods have been developed to site-speci¢cally that it charges the orthogonal suppressor tRNA with a introduce unnatural amino acids into proteins by suppres- desired unnatural amino acid, and none of the common sion of amber mutations with chemically acylated tRNAs 20 amino acids [3,4]. [1,2]. The ability to directly incorporate unnatural amino Several strategies have been developed to generate or- acids site-speci¢cally into proteins in living cells would thogonal suppressor tRNA^synthetase pairs in E. coli. greatly expand our ability to manipulate protein structure One involves destroying an existing E. coli tRNA's a¤nity and function as well as probe cellular processes. Our strat- toward its cognate synthetase (but not its ability to func- egy to expand the genetic code of Escherichia coli involves tion in translation) by mutating nucleotides at the tRNA^ the generation of a suppressor tRNA that is not acylated synthetase interface. A mutant synthetase is then evolved by any of the E. coli synthetases (orthogonal suppressor that uniquely recognizes the orthogonal tRNA. Such an tRNA), and a synthetase that does not acylate any E. coli orthogonal tRNA has been generated from E. coli Gln tRNA2 , but no mutant E. coli GlnRS has been evolved that charges the derived orthogonal tRNA more e¤ciently Gln than wild-type E. coli tRNA2 [4,5]. A second strategy 1 Present address: Department of Chemistry, The Scripps Research involves importing a tRNA^synthetase pair from another Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. organism into E. coli if cross-species aminoacylation is * Corresponding author. ine¤cient, and the suppressor tRNA derived from the E-mail address: [email protected] (P.G. Schultz). tRNA is not charged by E. coli synthetases. Two orthog- 1074-5521 / 01 / $ ^ see front matter ß 2001 Elsevier Science Ltd. All rights reserved. PII: S1074-5521(01)00063-1 884 Chemistry & Biology 8/9 (2001) 883^890 onal suppressor tRNA^synthetase pairs have been devel- To test the orthogonality of this suppressor tRNA in E. Gln oped based on the Saccharomyces cerevisiae tRNA2 ^ coli, an amber codon was introduced at a permissive site GlnRS and tRNAAsp^AspRS pairs [6,7]. Another ap- (Ala184) in the L-lactamase gene [6]. Those tRNAs that proach involves the use of a heterologous synthetase as can be charged by E. coli synthetases will suppress the the orthogonal synthetase but a mutant initiator tRNA amber codon and allow cells to live in the presence of Tyr of the same organism or a related organism as the orthog- ampicillin. The M. jannaschii tRNACUA suppresses the onal tRNA. Two pairs have been generated, a mutant amber codon in the L-lactamase gene with an IC50 value human initiator tRNA^E. coli GlnRS pair for use in S. of 56 Wg/ml ampicillin [9]. In contrast, the orthogonal fMet Gln Gln cerevisiae and a mutant E. coli initiator tRNA2 ^mutant tRNACUA derived from S. cerevisiae tRNA2 has an yeast TyrRS pair for use in E. coli [8]. IC50 of 21 Wg/ml ampicillin when tested in the same assay The development of additional orthogonal tRNA^syn- [6]. The IC50 for E. coli in the absence of any suppressor thetase pairs may allow the simultaneous incorporation of tRNA is 10 Wg/ml ampicillin. This result shows that the M. Tyr multiple unnatural amino acids into proteins. Moreover, jannaschii tRNACUA is a better substrate for E. coli syn- Gln di¡erent aminoacyl-tRNA synthetases may be better start- thetases than the tRNACUA. Consequently, if the M. jan- Tyr ing points for generating active sites with particular spe- naschii tRNACUA is used in vivo to deliver unnatural ami- ci¢cities, e.g. large hydrophobic vs. small hydrophilic ami- no acids into proteins in E. coli, it may also be mischarged no acids. We recently reported the generation of an with natural amino acids by E. coli synthetases, leading to Tyr orthogonal tRNACUA^TyrRS pair by expressing the heterogeneous amino acid incorporation. Tyr Methanococcus jannaschii tRNACUA^TyrRS pair in E. The improvement of the orthogonality of the M. janna- Tyr coli [9]. In comparison to the Gln and Asp orthogonal schii tRNACUA requires the introduction of `negative rec- pair, the TyrRS charges its cognate amber suppressor ognition determinants' to prevent recognition by endoge- tRNA with much higher e¤ciency. However, the nous E. coli synthetases. On the other hand, these Tyr tRNACUA derived from M. jannaschii is also a better sub- mutations should not strongly interfere with the tRNA's strate for the endogenous E. coli synthetases than the S. interaction with its cognate M. jannaschii TyrRS or the Gln Asp cerevisiae tRNACUA and tRNACUA. In order to improve ribosome. Since M. jannaschii TyrRS lacks most of the Tyr the utility of this tRNACUA^TyrRS pair, and to develop anticodon-binding domain [10], mutations introduced at additional such pairs, we have developed a general strat- the anticodon loop of the tRNA are expected to have a egy for selecting mutant tRNAs with enhanced orthogon- minimal e¡ect on TyrRS recognition. An anticodon-loop ality. Mutant suppressor tRNAs selected from libraries library with four randomized nucleotides was constructed Tyr derived from M. jannaschii tRNACUA have been generated to test this notion (Fig. 1). Given the various combina- and characterized. tions and locations of identity elements for various E. coli tRNAs, mutations at additional positions may increase the likelihood of ¢nding a mutant tRNA with the desired 2. Results and discussion properties. Thus, a second library containing mutations at nonconserved positions in all of the tRNA loops (all- 2.1. Suppressor tRNA library design and construction loop library) was also constructed (Fig. 1). Conserved nu- cleotides were not randomized so as to maintain the ter- Because of the complex nature of tRNA^synthetase in- tiary interactions that stabilize the `L'-shaped structure of teractions that are required to achieve a high degree of the tRNA [11,12]. Stem nucleotides were also not mutated ¢delity in protein translation, the rational design of or- since substitution of one such nucleotide requires a com- thogonal tRNA^synthetase pairs is di¤cult. Consequently, pensatory mutation. The 11 nucleotides (C16, C17, U17a, we have taken an approach that exploits the poor cross U20, C32, G37, A38, U45, U47, A59, and U60) were recognition of some interspecies tRNA^synthetase pairs, therefore randomized (Fig. 1). The theoretical size of this coupled with subsequent in vivo evolution of tRNAs library is 4.19U106, and a library with a size of 1.93U108 with enhanced orthogonality. The tRNATyr of M. janna- colony forming units was constructed to ensure complete schii, an archaebacterium, has di¡erent identity elements coverage of the mutant library. from those of E. coli tRNATyr. In particular, the E. coli tRNATyr has a G1C72pair in the acceptor stem while the 2.2. A general selection for orthogonal suppressor tRNAs in M. jannaschii tRNATyr has a C1G72pair. We have shown E. coli that an amber suppressor tRNA derived from M. janna- schii tRNATyr is not e¤ciently aminoacylated by the E. To select for a member of the M. jannaschii tRNA li- coli synthetases, but functions e¤ciently in protein trans- brary with enhanced orthogonality, we used a combina- lation in E. coli [9]. In addition, the M. jannaschii TyrRS, tion of negative and positive selections in the absence and which has only a minimalist anticodon-loop-binding do- presence of the cognate synthetase (Fig.
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