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Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs
Biochimie 95 (2013) 881e888
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Research paper
Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs Hong Tian 1, Danni Deng 1, Jie Huang, Dongning Yao, Xiaowei Xu, Xiangdong Gao* State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 April 2012 Accepted 7 December 2012 Available online 27 December 2012
Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a speciesspecific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNATyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blueewhite selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNATyr. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Orthogonality of tRNA Unnatural amino acid Screening system Expanding genetic code tRNA/synthetase pair
1. Introduction Expanding the genetic code is a recent approach that incorporates unnatural amino acids into proteins in vivo, with myriads of applications in the pharmaceutical and biotechnology industries [1]. As novel components in synthetic biology, proteins containing unnatural amino acids can be used in exploring protein structures and functions and in generating novel biological pharmaceuticals [2]. However, encoding unnatural amino acids in vivo requires a dedicated set of translational components, including a codon that specifies the unnatural amino acid, an orthogonal tRNA that can recognize the specific codon, and an orthogonal aminoacyltRNA synthetase (aaRS) that can charge the orthogonal tRNA with the unnatural amino acid. During protein synthesis in vivo,
Abbreviations: aaRS, aminoacyl-tRNA synthetase; EF-Tu, elongation factor Tu; Ec tRNATyr, E. coli tyrosyl tRNA; Mj tRNATyr, M. jannaschii tyrosyl tRNA; Sc tRNATrp, S. cerevisiae tryptophanyl tRNA; Mb tRNApyl, M. barkeri pyrrolysyl tRNA. * Corresponding author. Tel.: þ86 25 83271543; fax: þ86 25 83302827. E-mail address: [email protected] (X. Gao). 1 The first two authors contributed equally to this paper. 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2012.12.010
the orthogonal tRNA must not be aminoacylated by any of the host aaRS with natural amino acids. Similarly, the orthogonal aaRS must not transfer amino acids to host tRNAs; otherwise, the unnatural amino acid would be misincorporated throughout the proteome [3]. Orthogonal tRNA plays an important role in encoding unnatural amino acids in vivo because it evolves into a highly efficient amino acid carrier and activator during each stage of protein synthesis. The affinity of tRNA toward its cognate aaRS influences the efficiency of charging tRNA with an unnatural amino acid. The orthogonality of tRNA affects the fidelity of incorporation of unnatural amino acids into proteins, and its affinity for elongation factor Tu would thereby significantly influence the yield of proteins containing unnatural amino acids [4]. Moreover, the development of additional orthogonal tRNAs may allow the simultaneous incorporation of multiple unnatural amino acids into proteins. Several strategies were developed to generate orthogonal suppressor tRNA in Escherichia coli (Ec). The most popular strategy involves importing a tRNA/synthetase pair from another organism into Ec when the suppressor tRNA derived from the heterologous tRNA is not charged by Ec aaRS [5]. Using this procedure, several
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orthogonal suppressor tRNAs were generated, including the suppressor tRNAGln from Saccharomyces cerevisiae (Sc), the tRNAAsp from Sc, and the tRNAply from Methanosarcina barkeri (Mb) [6,7]. However, heterologous tRNAs are not always absolutely orthogonal to the endogenous aaRS. For example, the suppressor tRNATyr from Methanococcus jannaschii (Mj) could be charged by endogenous Ec tyrosyl tRNA synthetase (TyrRS) [8]. Similarly, the lysyl tRNA synthetase is responsible for misacylating the amber suppressor version of the yeast tryptophanyl tRNA [3]. To improve the orthogonality of such heterologous tRNA, mutant nucleotides need to be introduced into the tRNA to eliminate misacylation. Considering the complexity of tRNAeaaRS interactions, the strategy to achieve such an introduction is to build a random mutant library based on a rational design and then select cells expressing mutant tRNAs with enhanced orthogonality from this library. To this end, Schultz et al. [9] developed a screening system that could eliminate tRNAs with poor orthogonality from the pool by negative selection based on the suppression of amber nonsense mutations in the barnase gene. However, this strategy is not easy and is time consuming because the target tRNAs need to be excised from the negatively selected plasmids and then re-ligated into another plasmid for positive selection. Therefore, we developed a more convenient approach to identify functional suppressor tRNA mutants and expand further the potential utility of synthetic orthogonal pairs. In developing an amber suppressor tRNA, we observed a significantly slow growth rate of cells harboring suppressor tRNAs with poor orthogonality. Similar findings were reported in Sc strains that contain highly efficient amber (UAG) suppressors. These strains grow poorly on nutrient media. However, normal growth rates are observed when these strains lose the suppressors [10]. Moreover, the growth inhibition caused by suppressor tRNAs is species specific. The amber suppressor tRNAs derived from a species are usually toxic to the species itself but not to distantly related species, indicating a species-specific toxicity. In an unpublished thesis outcome, Pettersson [11] demonstrated that the Pseudomonas aeruginosa amber suppressor tRNATrp is toxic to P. aeruginosa but not to Ec. As expected, the P. aeruginosa amber suppressor tRNATrp could not be aminoacylated by Ec aaRS. Inspired by these findings, we explored an alternative strategy for screening orthogonal tRNAs based on the species-specific toxicity of suppressor tRNAs. In this work, we verified the species-specific toxicity of suppressor tRNAs using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaeans. We also verified whether the addition of a CCA terminus to the Mj tRNATyr mutant is important to its toxicity to Ec. In addition, we compared the toxicity of suppressor tRNAs toward the host using differing copy numbers. Subsequently, we developed a screening system derived from the high-copy-number plasmid pMD18T and obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNATyr. 2. Materials and methods 2.1. Strains and plasmids The Ec strain CSH108 was obtained from the Ec Genetic Stock Center (Strain # 8081). DH10B- and DH5a-competent cells were obtained from Takara (China). All restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA, USA). The vectors pACYC184 and pMD18-T were obtained from Takara (China), and pSB2K3 was obtained from the Biobrick parts registry at MIT (http://partsregistry.org/Main_Page). Oligonucleotides were obtained from Invitrogen (China). All buffers and chemicals were obtained from SigmaeAldrich (St. Louis, MO, USA) or Fisher Scientific (Waltham, MA, USA).
2.2. Synthesis of suppressor tRNA mutants We designed amber suppressor tRNA mutant genes based on previous reports. The oligonucleotides for tRNA gene synthesis (Supplementary Tables 1e4) were designed by DNAworks (http:// helixweb.nih.gov/dnaworks/) [12]. The mutant tRNA genes under the control of a proK promoter and terminator were synthesized using polymerase chain reaction (PCR)-based two-step DNA synthesis [13]. Briefly, 30 mM of 2#e7# oligonucleotides (inner primer) were added to a 500 mL microcentrifuge tube. The contents of the tubes were mixed by flicking and then pulsed spun in a microcentrifuge. A 0.5 mL aliquot of the oligonucleotide mixture was added to a thin-walled 500 mL PCR tube. Subsequently, 30 pmol of 1# and 8# oligonucleotides (outer primer) was added. Consequently, we added the PCR reaction mixture prepared as follows: 4 mL of 2.5 mM deoxyribonucleotide triphosphate was mixed with 5 mL of 10 buffer and 1 mL of Primestar polymerase, and then added with H2O to a final volume of 50 mL. PCR was conducted under the following conditions: 30 cycles of denaturation at 90 C for 30 s, annealing at 60 C for 45 s, and extension at 72 C for 50 s. A final extension was performed at 72 C for 5 min. 2.3. Growth curves of Ec DH10B containing different suppressor tRNAs Synthetic suppressor tRNA mutant genes were inserted into the plasmid pACYC184 to yield pAC-Ec tRNATyr, pAC-Mj tRNATyr, pAC-Sc tRNATrp, and pAC-Mb tRNAPyl. Then, the plasmids were transformed into DH10B-competent cells. One colony from each transformation was grown overnight in lysogeny broth (LB) medium supplemented with 50 mg/mL chloramphenicol. Cultures were diluted to an optical density at 600 nm (OD600) of 0.1. Then, 1 mL cultures was added into 100 mL LB medium. The cells were grown at 37 C for 10 h, and the absorption was read at 600 nm every 1 h. Similarly, Mj tRNATyrCCA and Sc tRNATrp-CCA were synthesized using the same procedures described above and then inserted into the plasmid pACYC184 to yield pAC-Mj tRNATyr -CCA and pAC-Sc tRNATrp-CCA. The growth curves of the cells were determined using the same procedures. 2.4. Construction of the pAC-tRNA, pMD-tRNA, and pSB2K3-tRNA plasmids The SC tRNA-CCA gene under the control of the proK promoter and terminator was synthesized using the same procedures described above. The synthesized gene fragments included the sites for the restriction enzymes Bam I and Xba I before the suppressor tRNA operon gene sequence. The sites for Spe I and Ava I were inserted behind the suppressor tRNA operon. The synthesized gene fragments were digested using the restriction enzymes Bam I and Ava I, gel purified, and ligated into the pre-digested pACYC184 vector to yield the plasmid pAC-tRNA. Similarly, the tRNA gene fragments were digested using the restriction enzymes Xba I and Spe I, and then ligated into the pre-digested pSB2K3 vector to yield the plasmid pSB2K3-tRNA. Given that pMD18-T is a commercial TA cloning vector, the synthesized gene fragments were amplified using Taq DNA polymerase and then ligated into pMD18-T to produce pMD-tRNA. 2.5. Effect of suppressor tRNA copy number on the growth rate of different Ec strains The plasmids pAC-tRNA and pMD-tRNA were transformed into DH10B, CSH108, and DH5a. The plasmids pACYC184 and pMD18-T were also transformed into the corresponding cells to serve as
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the control group. One colony from each transformation was grown overnight in LB medium. Cultures were diluted to an OD600 of 0.1. Then, 0.1 mL cultures were added into 10 mL LB medium in triplicate. The cells were grown at 37 C for 6 h, and the cell density (OD600) was measured. The results were reported as means SD. 2.6. Colony formation assay The plasmid pSB2K3-tRNA was transformed into DH10Bcompetent cells. Only freshly transformed cells were used. Numerous variant clones were observed when the earlier transformed cells were subcultured. The transformant cells were placed on two sets of LB plates (w103 cells for each set) containing kanamycin. One set contained 1 mmol/L isopropyl b-D-1-thiogalactopyranoside (IPTG). After an overnight incubation at 37 C, the colonies on each plate were counted. 2.7. Verification of the screening system To verify whether suppressor tRNAs with poor orthogonality were misaminoacylated in the host cells and could be eliminated by our screening system, Mj tRNATyr with a CCA terminus was inserted into pACYC184 and pMD18-T to yield pAC-tRNATyr-CCA and pMDtRNATyr-CCA, respectively. Then, pAC-tRNATyr-CCA and pMDtRNATyr-CCA were transformed into CSH108-competent cells. Transformed colonies were collected and suspended in 10 mL of LB medium. To clearly show the color of the colony that formed on the plate, colony spot assay was conducted. Aliquots (2 mL) of this mixture were spotted onto LB agar plates containing 50 mg/mL ampicillin, 1 mM IPTG, and 0.2 mg/mL 5-bromo-4-chloro-indolylb-D-galactopyranoside (X-gal). The spotted plates were incubated at 37 C overnight. To verify whether the blueewhite selection based on CSH108 could separate the orthogonal suppressor tRNA and non-functional suppressor tRNA, a plasmid pMD-mu-tRNATyr was constructed by inserting the mu-tRNATyr gene into pMD18-T. The mu-tRNATyr gene is a suppressor tRNA mutant derived from Mj tRNATyr, which has better orthogonality based on previous reports [14]. Then, the pACRS expressing TyrRS was co-transformed with pMD-mu-tRNATyr into CSH108. The plasmid pMD18-T without the mu-tRNATyr gene was also co-transformed with pAC-RS into CHS108 as a control. Then, the transformant cells were placed on LB agar plates containing 50 mg/mL ampicillin, 1 mM IPTG, and 0.2 mg/mL X-gal. 2.8. Construction and screening of the suppressor tRNA library The synthetic construct of the tRNA library was under the control of the proK promoter and terminator. The Fis binding site, naturally located upstream of the proK promoter, was also included in the sequence to enhance tRNA transcription (Supplementary Table 5). Then, the tRNA library was ligated into pMD18-T to construct pMDtRNAlib. pMD-tRNAlib was transformed into CSH108-competent cells containing pAC-NiRS, expressing the TyrRS mutant that could recognize p-nitrophenylalanine. Then, the transformant cells were grown on LB plates containing 25 mg/mL chloramphenicol, 50 mg/mL ampicillin, and 10 mg/mL p-nitrophenylalanine at 37 C for 10 h. When colonies with different colors (white, light blue, or deep blue) formed on the plates, the deep blue colonies were inoculated into LB liquid medium with 25 mg/mL chloramphenicol and 50 mg/mL ampicillin, and then grown to saturation at 37 C with shaking. Then, their pMD-tRNA plasmids were isolated using gel electrophoresis and retransformed into DH5a-competent cells. The positive transformants were inoculated into 5 mL LB medium, and the cultures were sent to a sequencing company.
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2.9. Chloramphenicol acetyltransferase (CAT) assay An amber codon was substituted for Asp112 in the CAT gene of pACYC184 to produce the plasmid pAC-Cm. The Mj TyrRS gene under the control of the Ec GlnRS promoter and terminator was digested using the restriction enzymes BamH I and Ava I and inserted into the predigested pAC-Cm to produce pAC-RS-Cm. The DH10B cells co-transformed with pMD-tRNA and pAC-Cm or pMDtRNA and pAC-Cm-RS were titrated against different chloramphenicol concentrations added to the growth medium. Cell growth was monitored by measuring OD600. 3. Results 3.1. Species-specific toxicity of the amber suppressor tRNA The amber suppressor tRNAs derived from Mj tRNATyr and Sc tRNATrp were selected as the research objects because of two reasons. First, they come from archaeans and eukaryotes, and both have species differences in relation to the host Ec; second, they could be misaminoacylated by Ec aaRS [3,15]. Mb tRNApyl was selected as a positive control for the orthogonal suppressor tRNA because it is a natural evolutionary suppressor tRNA that cannot be aminoacylated by any Ec aaRS [16]. Ec tRNATyr was selected as a negative control for the non-orthogonal suppressor tRNA. The tRNAs were synthesized by overlapping PCR using oligonucleotides. The anticodon of the tRNAs mutated to CAT (Table 1). These tRNAs were constructed into the plasmid pACYC184 under the control of the proK promoter and terminator, and then transformed into Ec DH10B-competent cells. The growth curve of the cells harboring suppressor tRNAs is shown in Fig. 1. As expected, the suppressor tRNAs from different sources exhibited speciesspecific toxicity toward the host. The Ec cells containing suppressor Ec tRNATyr mutants did not grow in the LB medium. Meanwhile, the growth rate of the cells containing the suppressor Mb tRNAPyl was similar to that of normal DH10B cells. The cells harboring the suppressor Sc tRNATrp or the suppressor Mj tRNATyr grew slower than the control group because these suppressor tRNAs could be charged by Ec aaRS, causing perturbation in the growth of host cells [3,15]. Moreover, the suppressor Mj tRNATyr demonstrated a greater inhibitory effect on the Ec cells than the suppressor Sc tRNATrp, suggesting that the suppressor Mj tRNATyr had poorer orthogonality. These results suggest that the speciesspecific toxicity of the suppressor tRNAs to the host depended on their orthogonality. Although the suppressors Mj tRNATyr and Sc tRNATrp inhibited the growth of their host cells, the mutations were not lethal. After 10 h of culture, the cells harboring the suppressor Sc tRNATrp achieved the same plateau as the control. To build an effective screening system, the inhibitory effect of the suppressor tRNAs should be enhanced.
Table 1 Sequences of suppressor tRNAs. Suppressor tRNAs
Sequences
Ec tRNATyr
50 -GGTGGGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATC TGCCGTCATCGACTTCGAAGGTTCGAATCCTTCCCCCACCACCA-30 50 -CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATC CGCATGGCAG GGGTTCAAATCCCCTCCGCCGGA-30 50 -GAAGCGGTGGCTCAATGGTAGAGCTTTCGACTCTAAATCGAA GGGTTGCAGGTTCAATTCCTGTCCGTTTCA-30 50 -GGGAACCTGATCATGTAGATCGAATGGACTCTAAATCCGTTTA GCCGGGTTAGATTCCCGGGGTTTCCGCCA-30
Mj tRNATyr Sc tRNATrp Mb tRNApyl
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Fig. 1. Growth curves of DH10B transformed with pAC-tRNA constructs. Constructs harbored a single copy of Mj tRNATyr, Sc tRNATrp, and Mb tRNAPyl between the proK promoter and the proK terminator, or pACYC with no tRNA (control). Cultures were grown at 37 C for 10 h in LB media supplemented with 25 mg/mL chloramphenicol and monitored by absorbance at 600 nm. Relative cell densities are indicated for 1 mL cultures and are shown as an average of 3 independent cultures.
The tRNA genes of both Mj tRNATyr and Sc tRNATrp lack the CCA sequence at their 30 end. The toxicities of the suppressor tRNA gene with and without the CCA sequence were compared. As shown in Fig. 2, the cells harboring the suppressor Sc tRNATrp gene with the CCA sequence at the 30 end (Sc tRNA-CCA) grew much slower than those harboring the suppressor Sc tRNATrp without this sequence. Moreover, the cells harboring the suppressor Mj tRNATyr with the CCA terminus did not grow. The results revealed that the suppressor tRNA gene with the CCA sequence at the 30 end presented greater toxicity toward the host, reiterating that the toxicity of suppressor tRNAs is a consequence of stop codon readthrough and is closely related to the orthogonality of tRNAs. 3.2. Effect of suppressor tRNA copy number on the host After verifying that the species-specific toxicity of the amber suppressor tRNAs toward the host depends on their orthogonality, we considered the possibility of building a negative screening system based on this species-specific toxicity. To verify whether a high amount of suppressor tRNA is lethal to the host cell, we inserted Sc tRNATrp with the CCA terminus into low- and highcopy-number plasmids. Then, the plasmids were transformed into three subtypes of Ec, namely, DH10B, CSH108, and DH5a.
Fig. 2. Growth curves of DH10B transformed with pAC-tRNA or pAC-tRNA-CCA constructs. Constructs harbored a single copy of Mj tRNATyr, Sc tRNATrp, Mj tRNATyrCCA, Sc tRNATrp-CCA, or pACYC with no tRNA (control). Cultures were grown at 37 C for 10 h in LB media supplemented with 25 mg/mL chloramphenicol and monitored by absorbance at 600 nm. Relative cell densities are indicated for 1 mL cultures and are shown as an average of 3 independent cultures.
Compared with the low-copy-number plasmid pACYC, Sc tRNACCA significantly inhibited the growth of the host cells when inserted into the high-copy-number plasmid pMD18-Tby. After 8 h of growth in the LB medium, the OD600 of the DH10B cells harboring pAC-tRNA was 94.3% of those harboring pACYC without the tRNA gene. Meanwhile, the optical density of the DH10B cells harboring pMD-tRNA was only 4.2% of the control cells. Similarly, the optical density of the CSH108 cells harboring pAC-tRNA was 87.8%, whereas that of the cells harboring pMD-tRNA was 1.3% of the control cells (Fig. 3). The growth inhibition by Sc tRNA-CCA in DH5a was weaker than that in other subtypes. This finding may be attributed to the fact that DH5a cells inherently contain the amber suppressor tRNA gene (glnV). This result suggests that the Ec subtypes containing the suppressor tRNA gene were not suitable hosts for negative screening. To confirm further whether a high-copy-number plasmid can be used in negative screening, the Sc tRNA-CCA gene was inserted into pSB2K3, an inducible copy number plasmid. This plasmid was then transformed into DH10B. Cells harboring pSB2K3-tRNA were placed on two sets of LB plates containing kanamycin (w103 cells for each set). One set contained 1 mmol/L IPTG. After overnight incubation, 3e5 colonies formed on the plates containing IPTG, whereas greater than 200 colonies formed on the plates without IPTG. When induced with IPTG, the copy number of pSB2K3 was greater than 100 per cell. Therefore, the cells harboring high copy numbers of the suppressor tRNAs with poor orthogonality could not form colonies on the LB plate. The result indicates that the high copy number of the suppressor tRNAs effectively eliminated the cells expressing the suppressor tRNAs with poor orthogonality. In theory, no colony should form on the plates containing IPTG, 3e5 colonies formed on the plates containing IPTG might have resulted from the inherent heterogeneity of the cells within a population. 3.3. Screening system for orthogonal suppressor tRNAs in Ec Based on the above results, we designed a screening system for orthogonal suppressor tRNAs. The tRNA mutation library was constructed using the high-copy-number plasmid pMD18-T and named pMD-tRNAlib. Cognate aaRS was constructed in the vector pACYC184, which is compatible with pMD18-T and named pAC-RS. The plasmid pMD-tRNAlib expressed the suppressor tRNA, and pAC-RS expressed aaRS. They were then co-transformed into the Ec strain CSH108 and screened for suppressor tRNA toxicity combined with blueewhite selection (Fig. 4).
Fig. 3. Effect of the copy number of suppressor tRNA on different subtypes of E. coli. The plasmids pAC-tRNA and pMD-tRNA were transformed into the DH10B, CSH108, and DH5a respectively. The pACYC184 and pMD18-T were also transformed into the corresponding cells to afford the control group. Each bar represents the mean of OD600 of triplicate samples SD.
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Fig. 4. Screening system for orthogonal suppressor tRNA. The plasmid pMD-tRNAlib expressed the suppressor tRNA while pAC-RS expressed aaRS; they were then co-transformed into the E. coli strain CSH108. When a member of the suppressor tRNA library is aminoacylated by endogenous E. coli aaRS, a large number of transcripts of suppressor tRNA would lead to host cell death. Meanwhile, as the strain CSH108 contains an episomal lacZ gene with an amber nonsense codon, if a suppressor tRNA is aminoacylated by its cognate aaRS, full-length b-galactosidase would be produced, resulting in a blue colony on the plate. Cells harboring nonfunctional tRNAs would yield a white colony due to in-frame translation termination.
To verify whether the suppressor tRNA with poor orthogonality misaminoacylated in the host cells and could be eliminated by our screening system, pAC-tRNATyr-CCA and pMD-tRNATyr-CCA were transformed into CSH108-competent cells. The strain CSH108 contained an episomal lacZ gene with an amber nonsense codon. If the suppressor Mj tRNATyr could be aminoacylated by Ec aaRS, then a full-length b-galactosidase would be produced, resulting in a blue lawn on the plate containing X-gal. As shown in Fig. 5A, a blue lawn formed on the plate, indicating that Mj tRNATyr could be aminoacylated by Ec aaRS. Our results are in accordance with those reported in a previous paper [17]. By contrast, the cells containing pMD-tRNATyr-CCA did not grow on the LB plate, so no lawn formed (Fig. 5B). Thus, cells containing the suppressor Mj tRNATyr with the CCA terminus could be eliminated by our screening system. To verify if the blueewhite selection using CSH108 could separate the orthogonal suppressor tRNA and non-functional suppressor tRNA, the plasmid pMD-mu-tRNATyr was constructed by inserting the mu-tRNATyr gene into pMD18-T. The mu-tRNATyr gene is a suppressor tRNA mutant derived from Mj tRNATyr, which has better orthogonality based on previous reports [14]. Then, the pACRS expressing TyrRS was co-transformed with pMD-mu-tRNATyr into CSH108. The plasmid pMD18-T without the mu-tRNATyr gene was also co-transformed with pAC-RS into CHS108 as a control. The CSH108 cells containing pAC-RS and pMD-Mj mu-tRNATyr formed blue colonies on the LB plate (Fig. 5C), whereas the CSH108 cells containing pAC-RS and pMD18-T formed white colonies on the plate (Fig. 5D). Considering that orthogonal suppressor tRNAs are used mainly in gene code expansion, Mj TyrRS mutants that can aminoacylate suppressor tRNAs with unnatural amino acids (e.g., p-acetylphenylalanine and p-nitrophenylalanine) were also verified. The CSH108 cells containing pAC-AcRS, expressing the TyrRS mutant that could recognize p-acetylphenylalanine, and pMD-Mj mutRNATyr formed blue colonies (Fig. 5E). Similarly, the CSH108 cells containing pAC-NiRS, expressing the TyrRS mutant that could
recognize p-nitrophenylalanine, and pMD-Mj mu-tRNATyr also formed blue colonies on the LB plate (Fig. 5F). 3.4. Suppressor tRNA library design, construction, and screening To demonstrate the effectiveness of our design strategy, we constructed the suppressor tRNA library derived from Mj tRNATyr. Based on a previous report, 11 non-conserved nucleotides (C16, C17, U17a, U20, C32, G37, A38, U45, U47, A59, and U60) were randomized to maximize the likelihood of identifying a mutant tRNA with high orthogonality. The conserved nucleotides were not randomized to maintain the tertiary interactions that stabilize the L-shaped structure of the tRNA (Fig. 6A) [9]. The suppressor tRNA library was screened using the strategy described earlier. As expected, when co-transformed with the TyrRS mutant specific to p-nitrophenylalanine, colonies with different colors (white, light blue, or deep blue) formed on the LB plate containing p-nitrophenylalanine (Fig. 6B). The white colonies indicate that the suppressor tRNA mutant in that colony was nonfunctional. A deep blue color implies that a full-length b-galactosidase was produced in the host cell, indicating that the suppressor tRNA mutant in that colony could be more effectively aminoacylated by the aaRS mutant specific to p-nitrophenylalanine. Therefore, three deep blue colonies were selected and cultured in the LB medium. The sequencing results of the three colonies revealed that three independent genes for suppressor tRNAs were isolated (Fig. 6C). To confirm the properties of the selected suppressor tRNAs, another conventional assay based on the suppression of an amber codon in the CAT gene was conducted [8]. In the CAT assay, an amber codon was substituted for Asp112 in the CAT gene of pACYC184 to produce pAC-Cm. The genes encoding Mj TyrRS were inserted into the pre-digested pAC-Cm to construct the plasmid pAC-RS-Cm. The three selected suppressor tRNAs were separated and retransformed into Ec DH10B with pAC-Cm or pAC-RS-Cm. Then,
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Fig. 5. Blueewhite selection of CSH108 containing orthogonal suppressor tRNA and aaRS mutant (A) CSH108 cells containing pMD-tRNATyr-CCA; (B) CSH108 cells containing pACtRNATyr-CCA; (C) CSH108 cells containing pAC-RS and pMD-Mj mu-tRNATyr; (D) CSH108 containing pAC-RS and pMD18-T; (E) CSH108 cells containing pAC-AcRS, expressing the tyrosyl-tRNA synthetase mutant that could recognize p-acetylphenylalanine, and pMD-Mj mu-tRNATyr on the plate containing p-acetylphenylalanine; (F) CSH108 cells containing pAC-NiRS, expressing the tyrosyl-tRNA synthetase mutant that could recognize p-nitrophenylalanine, and pMD-Mj mu-tRNATyr on the plate containing p-nitrophenylalanine.
the DH10B cells were cultured in the LB medium containing various concentrations of chloramphenicol. Considering that the amber codon was introduced in the chloramphenicol-resistant gene in pAC-RS-Cm and pAC-Cm, the chloramphenicol tolerance of DH10B
depended on the aminoacylation of the tRNA mutant. As expected, the chloramphenicol tolerance of DH10B containing pMD-tRNA and pAC-RS-Cm reached 80 mg/mL, whereas that of DH10B containing pMD-tRNA and pAC-Cm was lower than 20 mg/mL (Fig. 7). These
Fig. 6. Screening results of the suppressor tRNA library (A) tRNA library derived from M. jannaschii tRNATyr. Randomly mutated nucleotides (N), each mutant clone in an independent culture, are shaded in black. (B) CSH108 cells containing pAC-NiRS, expressing the tyrosyl-tRNA synthetase mutant that could recognize p-nitrophenylalanine, and pMDtRNAlib on the plate containing p-nitrophenylalanine; (C) DNA sequences of mutant suppressor tRNAs selected from the suppressor tRNA library.
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Fig. 7. CAT assay results of selected suppressor tRNAs. Dark gray line represented the chloramphenicol tolerance of DH10B containing the pMD-tRNA which expresses the selected suppressor tRNA and pAC-RS-Cm which express the M. jannaschii tyrosyl tRNA synthetase. Light gray line represented the chloramphenicol tolerance of DH10B containing the pMD-tRNA and pAC-Cm that without the M. jannaschii tyrosyl tRNA synthetase gene.
results indicate that the selected suppressor tRNAs could be aminoacylated by Mj TyrRS but not by any Ec aaRS. Thus, the selected suppressor tRNAs were orthogonal based on the CAT assay. 4. Discussion The primary goal of our study is to establish a high-throughput screening approach for obtaining orthogonal suppressor tRNAs based on their species-specific toxicity. Therefore, we first verified if the species-specific toxicity of an amber suppressor tRNA toward the host depends on its orthogonality. The suppressor tRNATyr from Ec completely inhibited the growth of Ec cells under the conditions used, which may have resulted in the production of abnormal proteins in bacteria via readthrough of a stop codon by the suppressor tRNAs. Although the amber codon TAG is the least used nonsense triplet in Ec (w9%), 320 genes in Ec terminate in the UAG codon, including 44 essential genes [18]. By contrast, Mb tRNAPyl is
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a natural evolutionary suppressor tRNA that can be aminoacylated by pyrrolysyl-tRNA synthetase (PylRS). When transformed into Ec, Mb PylRS and tRNAPyl function as an orthogonal pair in vivo [19]. The special structure of tRNAPyl renders it incapable of aminoacylation by any Ec aaRS. Therefore, tRNAPyl is not toxic to the Ec host. In contrast to tRNAPyl, the suppressor Mj tRNATyr is a substrate for the endogenous Ec synthetases [15]. Our results also showed that cells harboring the suppressor Mj tRNATyr conduct a translational readthrough of the b-galactosidase gene containing the amber codon mutant. Hence, the poor orthogonality leads to a wild suppressor Mj tRNATyr that can be charged by the Ec endogenous aaRS and cause perturbation to the growth of the host cells. Similarly, Sc tRNATrp was proven to be a substrate for Ec lysyl tRNA synthetase [3], Therefore, the cells harboring Sc tRNATrp grew slower than the control group. The tRNA genes of both Mj tRNATyr and Sc tRNATrp lack the CCA sequence at their 30 end. These tRNAs need the addition of the CCA sequence in the maturation process by ATP (cytidine triphosphate):tRNA nucleotidyl transferase, which is present in archaeans, bacteria, and eukaryotes, as described by Woese [20]. When the complete CCA sequence is absent in the tRNA precursor, as in the cases of Mj tRNATyr and Sc tRNATrp, RNase BN removes the extra nucleotides from the 30 terminus of the precursor RNA and then the CCA 30 -OH terminus could be added by the CCA enzyme [21]. As a result, the tRNA without the CCA sequence at the 30 end requires more steps to mature before it can be aminoacylated, thereby inhibiting the growth of the host cells, but not being lethal to the host cells. A recent kinetic report has revealed that the Ec CCA enzyme has the innate ability to discriminate against tRNA backbone damage at each step of the CCA synthesis [22], leading to the inactivation of extraneous suppressor tRNA mutants. These results revealed that the suppressor tRNA gene with a CCA sequence at the 30 end presented greater toxicity to the host. Thus, the toxicity of suppressor tRNAs is a consequence of stop codon readthrough and is closely related to the orthogonality of tRNAs. A prerequisite to building a negative screening system is effectively eliminating tRNA mutants with poor orthogonality, which means that the toxicity of the suppressor tRNAs must be severe enough to kill the host. Our results revealed that the suppressor tRNAs inserted into high-copy-number plasmids exhibited stronger effects in inhibiting host growth. The readthrough event may have been a result of competition between release factors and suppressor tRNAs for the amber codon. Thus, increasing the copy numbers of suppressor tRNAs increases the suppression of all amber codons in the cell, enhancing the readthrough of stop codons on chromosomal genes [23]. To confirm further the effect of copy numbers on the host, an inducible copy number plasmid, pSB2K3, was used in this study. The pSB2K3 plasmid contains the P1 lytic replication origin, which is under the control of a lacI-regulated promoter. When induced with IPTG, P1 lytic replication proteins are produced. Thus, the P1 lytic replication origin is activated, raising the copy number to very high levels (http://partsregistry. org/Part:pSB2K3). The cells harboring pSB-tRNA formed less colonies on the plate containing IPTG, demonstrating that suppressor tRNAs in high-copy-number plasmids could effectively eliminate tRNA mutants with poor orthogonality. Therefore, this high-copynumber plasmid pMD18-T was selected as the screening vector. Another advantage of pMD18-T is its convenience for sequencing because it is derived from pUC18 and its sequencing primers are widely commercialized. Based on these results, we designed a screening system for orthogonal suppressor tRNAs. A suppressor tRNA mutant library was inserted into pMD18-T to form negative screening components. When a member of the suppressor tRNA library was aminoacylated by endogenous Ec aaRS (i.e., not orthogonal to Ec aaRS),
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many transcripts of suppressor tRNAs would lead to host cell death. Only cells harboring orthogonal tRNAs or non-functional tRNAs would survive. Furthermore, given that the strain CSH108 contained an episomal lacZ gene with an amber nonsense codon, a fulllength b-galactosidase was produced upon the aminoacylation of a suppressor tRNA by its cognate aaRS. Thus, a blue colony formed on the plate containing the galactoside analog X-gal. By contrast, the cells harboring non-functional tRNAs yielded a white colony because of the in-frame translation termination. In addition, the colonies presented various shades of blue, suggesting that the suppressor tRNA mutant in the deep blue colony may have been more effectively aminoacylated by its cognate aaRS than that in the light blue colony. Thus, by combining the analysis on the toxicity of the suppressor tRNAs toward the host with blueewhite detection, we developed a convenient screening system for identifying orthogonal suppressor tRNAs, which could serve as a general platform for generating tRNA/aaRS pairs. Acknowledgments This work has been supported by the Chinese National Natural Science Foundation (31200694 and 81273426), Important National Science & Technology Specific Projects (2009ZX09102-224), National Fund for Fostering Talents of Basic Science (J0630858) and Qing Lan Projects. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biochi.2012.12.010. References [1] Q. Wang, A.R. Parrish, L. Wang, Expanding the genetic code for biological studies, Chem. Biol. 16 (2009) 323e336. [2] J. Xie, P.G. Schultz, A chemical toolkit for proteins e an expanded genetic code, Nat. Rev. Mol. Cell. Biol. 7 (2006) 775e782. [3] R.A. Hughes, A.D. Ellington, Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA, Nucleic Acids Res. 38 (2010) 6813e6830. [4] J.M. Schrader, S.J. Chapman, O.C. Uhlenbeck, Understanding the sequence specificity of tRNA binding to elongation factor Tu using tRNA mutagenesis, J. Mol. Biol. 386 (2009) 1255e1264.
[5] D.R. Liu, P.G. Schultz, Progress toward the evolution of an organism with an expanded genetic code, Proc. Natl. Acad. Sci. U S A 96 (1999) 4780e4785. [6] D.R. Liu, T.J. Magliery, M. Pastrnak, P.G. Schultz, Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo, Proc. Natl. Acad. Sci. U S A 94 (1997) 10092e10097. [7] A.K. Kowal, C. Kohrer, U.L. RajBhandary, Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria, Proc. Natl. Acad. Sci. U S A 98 (2001) 2268e2273. [8] M. Pastrnak, T.J. Magliery, P.G. Schultz, A new orthogonal suppressor tRNA/ aminoacyl-tRNA synthetase pair for evolving an organism with an expanded genetic code, Helv. Chim. Acta 83 (2000) 2277e2286. [9] L. Wang, P.G. Schultz, A general approach for the generation of orthogonal tRNAs, Chem. Biol. 8 (2001) 883e890. [10] S.W. Liebman, F. Sherman, Inhibition of growth by amber suppressors in yeast, Genetics 82 (1976) 233e249. [11] B.M.F. Pettersson, tRNA Gene Structures in Bacteria, Uppsala University, 2009, pp. 39e41. [12] D.M. Hoover, J. Lubkowski, DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis, Nucleic Acids Res. 30 (2002) e43. [13] A.S. Xiong, Q.H. Yao, R.H. Peng, H. Duan, X. Li, H.Q. Fan, Z.M. Cheng, Y. Li, PCR-based accurate synthesis of long DNA sequences, Nat. Protoc. 1 (2006) 791e797. [14] J. Guo, C.E. Melancon 3rd, H.S. Lee, D. Groff, P.G. Schultz, Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids, Angew. Chem. Int. Ed. Engl. 48 (2009) 9148e9151. [15] L. Wang, T.J. Magliery, D.R. Liu, P.G. Schultz, A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins, J. Am. Chem. Soc. 122 (2000) 5010e5011. [16] K. Nozawa, P. O’Donoghue, S. Gundllapalli, Y. Araiso, R. Ishitani, T. Umehara, D. Söll, O. Nureki, Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality, Nature 457 (2009) 1163e1167. [17] F.R. Blattner, G. Plunkett 3rd, C.A. Bloch, N.T. Perna, V. Burland, M. Riley, J. Collado-Vides, J.D. Glasner, C.K. Rode, G.F. Mayhew, J. Gregor, N.W. Davis, H.A. Kirkpatrick, M.A. Goeden, D.J. Rose, B. Mau, Y. Shao, The complete genome sequence of Escherichia coli K-12, Science 277 (1997) 1453e1462. [18] K. Wang, H. Neumann, S.Y. Peak-Chew, J.W. Chin, Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion, Nat. Biotechnol. 25 (2007) 770e777. [19] S.M. Kuhn, M. Rubini, M. Fuhrmann, I. Theobald, A. Skerra, Engineering of an orthogonal aminoacyl-tRNA synthetase for efficient incorporation of the nonnatural amino acid O-methyl-L-tyrosine using fluorescence-based bacterial cell sorting, J. Mol. Biol. 404 (2010) 70e87. [20] C.R. Woese, G.E. Fox, Phylogenetic structure of the prokaryotic domain: the primary kingdoms, Proc. Natl. Acad. Sci. U S A 74 (1977) 5088e5090. [21] G.P. Mazzara, G. Plunkett 3rd, W.H. McClain, DNA sequence of the transfer RNA region of bacteriophage T4: implications for transfer RNA synthesis, Proc. Natl. Acad. Sci. U S A 78 (1981) 889e892. [22] Y.M. Hou, CCA addition to tRNA: implications for tRNA quality control, IUBMB Life 62 (2010) 251e260. [23] A.A. Korostelev, Structural aspects of translation termination on the ribosome, RNA 17 (2010) 1409e1421.
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Research paper
Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs Hong Tian 1, Danni Deng 1, Jie Huang, Dongning Yao, Xiaowei Xu, Xiangdong Gao* State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 April 2012 Accepted 7 December 2012 Available online 27 December 2012
Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a speciesspecific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNATyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blueewhite selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNATyr. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Orthogonality of tRNA Unnatural amino acid Screening system Expanding genetic code tRNA/synthetase pair
1. Introduction Expanding the genetic code is a recent approach that incorporates unnatural amino acids into proteins in vivo, with myriads of applications in the pharmaceutical and biotechnology industries [1]. As novel components in synthetic biology, proteins containing unnatural amino acids can be used in exploring protein structures and functions and in generating novel biological pharmaceuticals [2]. However, encoding unnatural amino acids in vivo requires a dedicated set of translational components, including a codon that specifies the unnatural amino acid, an orthogonal tRNA that can recognize the specific codon, and an orthogonal aminoacyltRNA synthetase (aaRS) that can charge the orthogonal tRNA with the unnatural amino acid. During protein synthesis in vivo,
Abbreviations: aaRS, aminoacyl-tRNA synthetase; EF-Tu, elongation factor Tu; Ec tRNATyr, E. coli tyrosyl tRNA; Mj tRNATyr, M. jannaschii tyrosyl tRNA; Sc tRNATrp, S. cerevisiae tryptophanyl tRNA; Mb tRNApyl, M. barkeri pyrrolysyl tRNA. * Corresponding author. Tel.: þ86 25 83271543; fax: þ86 25 83302827. E-mail address: [email protected] (X. Gao). 1 The first two authors contributed equally to this paper. 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2012.12.010
the orthogonal tRNA must not be aminoacylated by any of the host aaRS with natural amino acids. Similarly, the orthogonal aaRS must not transfer amino acids to host tRNAs; otherwise, the unnatural amino acid would be misincorporated throughout the proteome [3]. Orthogonal tRNA plays an important role in encoding unnatural amino acids in vivo because it evolves into a highly efficient amino acid carrier and activator during each stage of protein synthesis. The affinity of tRNA toward its cognate aaRS influences the efficiency of charging tRNA with an unnatural amino acid. The orthogonality of tRNA affects the fidelity of incorporation of unnatural amino acids into proteins, and its affinity for elongation factor Tu would thereby significantly influence the yield of proteins containing unnatural amino acids [4]. Moreover, the development of additional orthogonal tRNAs may allow the simultaneous incorporation of multiple unnatural amino acids into proteins. Several strategies were developed to generate orthogonal suppressor tRNA in Escherichia coli (Ec). The most popular strategy involves importing a tRNA/synthetase pair from another organism into Ec when the suppressor tRNA derived from the heterologous tRNA is not charged by Ec aaRS [5]. Using this procedure, several
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orthogonal suppressor tRNAs were generated, including the suppressor tRNAGln from Saccharomyces cerevisiae (Sc), the tRNAAsp from Sc, and the tRNAply from Methanosarcina barkeri (Mb) [6,7]. However, heterologous tRNAs are not always absolutely orthogonal to the endogenous aaRS. For example, the suppressor tRNATyr from Methanococcus jannaschii (Mj) could be charged by endogenous Ec tyrosyl tRNA synthetase (TyrRS) [8]. Similarly, the lysyl tRNA synthetase is responsible for misacylating the amber suppressor version of the yeast tryptophanyl tRNA [3]. To improve the orthogonality of such heterologous tRNA, mutant nucleotides need to be introduced into the tRNA to eliminate misacylation. Considering the complexity of tRNAeaaRS interactions, the strategy to achieve such an introduction is to build a random mutant library based on a rational design and then select cells expressing mutant tRNAs with enhanced orthogonality from this library. To this end, Schultz et al. [9] developed a screening system that could eliminate tRNAs with poor orthogonality from the pool by negative selection based on the suppression of amber nonsense mutations in the barnase gene. However, this strategy is not easy and is time consuming because the target tRNAs need to be excised from the negatively selected plasmids and then re-ligated into another plasmid for positive selection. Therefore, we developed a more convenient approach to identify functional suppressor tRNA mutants and expand further the potential utility of synthetic orthogonal pairs. In developing an amber suppressor tRNA, we observed a significantly slow growth rate of cells harboring suppressor tRNAs with poor orthogonality. Similar findings were reported in Sc strains that contain highly efficient amber (UAG) suppressors. These strains grow poorly on nutrient media. However, normal growth rates are observed when these strains lose the suppressors [10]. Moreover, the growth inhibition caused by suppressor tRNAs is species specific. The amber suppressor tRNAs derived from a species are usually toxic to the species itself but not to distantly related species, indicating a species-specific toxicity. In an unpublished thesis outcome, Pettersson [11] demonstrated that the Pseudomonas aeruginosa amber suppressor tRNATrp is toxic to P. aeruginosa but not to Ec. As expected, the P. aeruginosa amber suppressor tRNATrp could not be aminoacylated by Ec aaRS. Inspired by these findings, we explored an alternative strategy for screening orthogonal tRNAs based on the species-specific toxicity of suppressor tRNAs. In this work, we verified the species-specific toxicity of suppressor tRNAs using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaeans. We also verified whether the addition of a CCA terminus to the Mj tRNATyr mutant is important to its toxicity to Ec. In addition, we compared the toxicity of suppressor tRNAs toward the host using differing copy numbers. Subsequently, we developed a screening system derived from the high-copy-number plasmid pMD18T and obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNATyr. 2. Materials and methods 2.1. Strains and plasmids The Ec strain CSH108 was obtained from the Ec Genetic Stock Center (Strain # 8081). DH10B- and DH5a-competent cells were obtained from Takara (China). All restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA, USA). The vectors pACYC184 and pMD18-T were obtained from Takara (China), and pSB2K3 was obtained from the Biobrick parts registry at MIT (http://partsregistry.org/Main_Page). Oligonucleotides were obtained from Invitrogen (China). All buffers and chemicals were obtained from SigmaeAldrich (St. Louis, MO, USA) or Fisher Scientific (Waltham, MA, USA).
2.2. Synthesis of suppressor tRNA mutants We designed amber suppressor tRNA mutant genes based on previous reports. The oligonucleotides for tRNA gene synthesis (Supplementary Tables 1e4) were designed by DNAworks (http:// helixweb.nih.gov/dnaworks/) [12]. The mutant tRNA genes under the control of a proK promoter and terminator were synthesized using polymerase chain reaction (PCR)-based two-step DNA synthesis [13]. Briefly, 30 mM of 2#e7# oligonucleotides (inner primer) were added to a 500 mL microcentrifuge tube. The contents of the tubes were mixed by flicking and then pulsed spun in a microcentrifuge. A 0.5 mL aliquot of the oligonucleotide mixture was added to a thin-walled 500 mL PCR tube. Subsequently, 30 pmol of 1# and 8# oligonucleotides (outer primer) was added. Consequently, we added the PCR reaction mixture prepared as follows: 4 mL of 2.5 mM deoxyribonucleotide triphosphate was mixed with 5 mL of 10 buffer and 1 mL of Primestar polymerase, and then added with H2O to a final volume of 50 mL. PCR was conducted under the following conditions: 30 cycles of denaturation at 90 C for 30 s, annealing at 60 C for 45 s, and extension at 72 C for 50 s. A final extension was performed at 72 C for 5 min. 2.3. Growth curves of Ec DH10B containing different suppressor tRNAs Synthetic suppressor tRNA mutant genes were inserted into the plasmid pACYC184 to yield pAC-Ec tRNATyr, pAC-Mj tRNATyr, pAC-Sc tRNATrp, and pAC-Mb tRNAPyl. Then, the plasmids were transformed into DH10B-competent cells. One colony from each transformation was grown overnight in lysogeny broth (LB) medium supplemented with 50 mg/mL chloramphenicol. Cultures were diluted to an optical density at 600 nm (OD600) of 0.1. Then, 1 mL cultures was added into 100 mL LB medium. The cells were grown at 37 C for 10 h, and the absorption was read at 600 nm every 1 h. Similarly, Mj tRNATyrCCA and Sc tRNATrp-CCA were synthesized using the same procedures described above and then inserted into the plasmid pACYC184 to yield pAC-Mj tRNATyr -CCA and pAC-Sc tRNATrp-CCA. The growth curves of the cells were determined using the same procedures. 2.4. Construction of the pAC-tRNA, pMD-tRNA, and pSB2K3-tRNA plasmids The SC tRNA-CCA gene under the control of the proK promoter and terminator was synthesized using the same procedures described above. The synthesized gene fragments included the sites for the restriction enzymes Bam I and Xba I before the suppressor tRNA operon gene sequence. The sites for Spe I and Ava I were inserted behind the suppressor tRNA operon. The synthesized gene fragments were digested using the restriction enzymes Bam I and Ava I, gel purified, and ligated into the pre-digested pACYC184 vector to yield the plasmid pAC-tRNA. Similarly, the tRNA gene fragments were digested using the restriction enzymes Xba I and Spe I, and then ligated into the pre-digested pSB2K3 vector to yield the plasmid pSB2K3-tRNA. Given that pMD18-T is a commercial TA cloning vector, the synthesized gene fragments were amplified using Taq DNA polymerase and then ligated into pMD18-T to produce pMD-tRNA. 2.5. Effect of suppressor tRNA copy number on the growth rate of different Ec strains The plasmids pAC-tRNA and pMD-tRNA were transformed into DH10B, CSH108, and DH5a. The plasmids pACYC184 and pMD18-T were also transformed into the corresponding cells to serve as
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the control group. One colony from each transformation was grown overnight in LB medium. Cultures were diluted to an OD600 of 0.1. Then, 0.1 mL cultures were added into 10 mL LB medium in triplicate. The cells were grown at 37 C for 6 h, and the cell density (OD600) was measured. The results were reported as means SD. 2.6. Colony formation assay The plasmid pSB2K3-tRNA was transformed into DH10Bcompetent cells. Only freshly transformed cells were used. Numerous variant clones were observed when the earlier transformed cells were subcultured. The transformant cells were placed on two sets of LB plates (w103 cells for each set) containing kanamycin. One set contained 1 mmol/L isopropyl b-D-1-thiogalactopyranoside (IPTG). After an overnight incubation at 37 C, the colonies on each plate were counted. 2.7. Verification of the screening system To verify whether suppressor tRNAs with poor orthogonality were misaminoacylated in the host cells and could be eliminated by our screening system, Mj tRNATyr with a CCA terminus was inserted into pACYC184 and pMD18-T to yield pAC-tRNATyr-CCA and pMDtRNATyr-CCA, respectively. Then, pAC-tRNATyr-CCA and pMDtRNATyr-CCA were transformed into CSH108-competent cells. Transformed colonies were collected and suspended in 10 mL of LB medium. To clearly show the color of the colony that formed on the plate, colony spot assay was conducted. Aliquots (2 mL) of this mixture were spotted onto LB agar plates containing 50 mg/mL ampicillin, 1 mM IPTG, and 0.2 mg/mL 5-bromo-4-chloro-indolylb-D-galactopyranoside (X-gal). The spotted plates were incubated at 37 C overnight. To verify whether the blueewhite selection based on CSH108 could separate the orthogonal suppressor tRNA and non-functional suppressor tRNA, a plasmid pMD-mu-tRNATyr was constructed by inserting the mu-tRNATyr gene into pMD18-T. The mu-tRNATyr gene is a suppressor tRNA mutant derived from Mj tRNATyr, which has better orthogonality based on previous reports [14]. Then, the pACRS expressing TyrRS was co-transformed with pMD-mu-tRNATyr into CSH108. The plasmid pMD18-T without the mu-tRNATyr gene was also co-transformed with pAC-RS into CHS108 as a control. Then, the transformant cells were placed on LB agar plates containing 50 mg/mL ampicillin, 1 mM IPTG, and 0.2 mg/mL X-gal. 2.8. Construction and screening of the suppressor tRNA library The synthetic construct of the tRNA library was under the control of the proK promoter and terminator. The Fis binding site, naturally located upstream of the proK promoter, was also included in the sequence to enhance tRNA transcription (Supplementary Table 5). Then, the tRNA library was ligated into pMD18-T to construct pMDtRNAlib. pMD-tRNAlib was transformed into CSH108-competent cells containing pAC-NiRS, expressing the TyrRS mutant that could recognize p-nitrophenylalanine. Then, the transformant cells were grown on LB plates containing 25 mg/mL chloramphenicol, 50 mg/mL ampicillin, and 10 mg/mL p-nitrophenylalanine at 37 C for 10 h. When colonies with different colors (white, light blue, or deep blue) formed on the plates, the deep blue colonies were inoculated into LB liquid medium with 25 mg/mL chloramphenicol and 50 mg/mL ampicillin, and then grown to saturation at 37 C with shaking. Then, their pMD-tRNA plasmids were isolated using gel electrophoresis and retransformed into DH5a-competent cells. The positive transformants were inoculated into 5 mL LB medium, and the cultures were sent to a sequencing company.
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2.9. Chloramphenicol acetyltransferase (CAT) assay An amber codon was substituted for Asp112 in the CAT gene of pACYC184 to produce the plasmid pAC-Cm. The Mj TyrRS gene under the control of the Ec GlnRS promoter and terminator was digested using the restriction enzymes BamH I and Ava I and inserted into the predigested pAC-Cm to produce pAC-RS-Cm. The DH10B cells co-transformed with pMD-tRNA and pAC-Cm or pMDtRNA and pAC-Cm-RS were titrated against different chloramphenicol concentrations added to the growth medium. Cell growth was monitored by measuring OD600. 3. Results 3.1. Species-specific toxicity of the amber suppressor tRNA The amber suppressor tRNAs derived from Mj tRNATyr and Sc tRNATrp were selected as the research objects because of two reasons. First, they come from archaeans and eukaryotes, and both have species differences in relation to the host Ec; second, they could be misaminoacylated by Ec aaRS [3,15]. Mb tRNApyl was selected as a positive control for the orthogonal suppressor tRNA because it is a natural evolutionary suppressor tRNA that cannot be aminoacylated by any Ec aaRS [16]. Ec tRNATyr was selected as a negative control for the non-orthogonal suppressor tRNA. The tRNAs were synthesized by overlapping PCR using oligonucleotides. The anticodon of the tRNAs mutated to CAT (Table 1). These tRNAs were constructed into the plasmid pACYC184 under the control of the proK promoter and terminator, and then transformed into Ec DH10B-competent cells. The growth curve of the cells harboring suppressor tRNAs is shown in Fig. 1. As expected, the suppressor tRNAs from different sources exhibited speciesspecific toxicity toward the host. The Ec cells containing suppressor Ec tRNATyr mutants did not grow in the LB medium. Meanwhile, the growth rate of the cells containing the suppressor Mb tRNAPyl was similar to that of normal DH10B cells. The cells harboring the suppressor Sc tRNATrp or the suppressor Mj tRNATyr grew slower than the control group because these suppressor tRNAs could be charged by Ec aaRS, causing perturbation in the growth of host cells [3,15]. Moreover, the suppressor Mj tRNATyr demonstrated a greater inhibitory effect on the Ec cells than the suppressor Sc tRNATrp, suggesting that the suppressor Mj tRNATyr had poorer orthogonality. These results suggest that the speciesspecific toxicity of the suppressor tRNAs to the host depended on their orthogonality. Although the suppressors Mj tRNATyr and Sc tRNATrp inhibited the growth of their host cells, the mutations were not lethal. After 10 h of culture, the cells harboring the suppressor Sc tRNATrp achieved the same plateau as the control. To build an effective screening system, the inhibitory effect of the suppressor tRNAs should be enhanced.
Table 1 Sequences of suppressor tRNAs. Suppressor tRNAs
Sequences
Ec tRNATyr
50 -GGTGGGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATC TGCCGTCATCGACTTCGAAGGTTCGAATCCTTCCCCCACCACCA-30 50 -CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATC CGCATGGCAG GGGTTCAAATCCCCTCCGCCGGA-30 50 -GAAGCGGTGGCTCAATGGTAGAGCTTTCGACTCTAAATCGAA GGGTTGCAGGTTCAATTCCTGTCCGTTTCA-30 50 -GGGAACCTGATCATGTAGATCGAATGGACTCTAAATCCGTTTA GCCGGGTTAGATTCCCGGGGTTTCCGCCA-30
Mj tRNATyr Sc tRNATrp Mb tRNApyl
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Fig. 1. Growth curves of DH10B transformed with pAC-tRNA constructs. Constructs harbored a single copy of Mj tRNATyr, Sc tRNATrp, and Mb tRNAPyl between the proK promoter and the proK terminator, or pACYC with no tRNA (control). Cultures were grown at 37 C for 10 h in LB media supplemented with 25 mg/mL chloramphenicol and monitored by absorbance at 600 nm. Relative cell densities are indicated for 1 mL cultures and are shown as an average of 3 independent cultures.
The tRNA genes of both Mj tRNATyr and Sc tRNATrp lack the CCA sequence at their 30 end. The toxicities of the suppressor tRNA gene with and without the CCA sequence were compared. As shown in Fig. 2, the cells harboring the suppressor Sc tRNATrp gene with the CCA sequence at the 30 end (Sc tRNA-CCA) grew much slower than those harboring the suppressor Sc tRNATrp without this sequence. Moreover, the cells harboring the suppressor Mj tRNATyr with the CCA terminus did not grow. The results revealed that the suppressor tRNA gene with the CCA sequence at the 30 end presented greater toxicity toward the host, reiterating that the toxicity of suppressor tRNAs is a consequence of stop codon readthrough and is closely related to the orthogonality of tRNAs. 3.2. Effect of suppressor tRNA copy number on the host After verifying that the species-specific toxicity of the amber suppressor tRNAs toward the host depends on their orthogonality, we considered the possibility of building a negative screening system based on this species-specific toxicity. To verify whether a high amount of suppressor tRNA is lethal to the host cell, we inserted Sc tRNATrp with the CCA terminus into low- and highcopy-number plasmids. Then, the plasmids were transformed into three subtypes of Ec, namely, DH10B, CSH108, and DH5a.
Fig. 2. Growth curves of DH10B transformed with pAC-tRNA or pAC-tRNA-CCA constructs. Constructs harbored a single copy of Mj tRNATyr, Sc tRNATrp, Mj tRNATyrCCA, Sc tRNATrp-CCA, or pACYC with no tRNA (control). Cultures were grown at 37 C for 10 h in LB media supplemented with 25 mg/mL chloramphenicol and monitored by absorbance at 600 nm. Relative cell densities are indicated for 1 mL cultures and are shown as an average of 3 independent cultures.
Compared with the low-copy-number plasmid pACYC, Sc tRNACCA significantly inhibited the growth of the host cells when inserted into the high-copy-number plasmid pMD18-Tby. After 8 h of growth in the LB medium, the OD600 of the DH10B cells harboring pAC-tRNA was 94.3% of those harboring pACYC without the tRNA gene. Meanwhile, the optical density of the DH10B cells harboring pMD-tRNA was only 4.2% of the control cells. Similarly, the optical density of the CSH108 cells harboring pAC-tRNA was 87.8%, whereas that of the cells harboring pMD-tRNA was 1.3% of the control cells (Fig. 3). The growth inhibition by Sc tRNA-CCA in DH5a was weaker than that in other subtypes. This finding may be attributed to the fact that DH5a cells inherently contain the amber suppressor tRNA gene (glnV). This result suggests that the Ec subtypes containing the suppressor tRNA gene were not suitable hosts for negative screening. To confirm further whether a high-copy-number plasmid can be used in negative screening, the Sc tRNA-CCA gene was inserted into pSB2K3, an inducible copy number plasmid. This plasmid was then transformed into DH10B. Cells harboring pSB2K3-tRNA were placed on two sets of LB plates containing kanamycin (w103 cells for each set). One set contained 1 mmol/L IPTG. After overnight incubation, 3e5 colonies formed on the plates containing IPTG, whereas greater than 200 colonies formed on the plates without IPTG. When induced with IPTG, the copy number of pSB2K3 was greater than 100 per cell. Therefore, the cells harboring high copy numbers of the suppressor tRNAs with poor orthogonality could not form colonies on the LB plate. The result indicates that the high copy number of the suppressor tRNAs effectively eliminated the cells expressing the suppressor tRNAs with poor orthogonality. In theory, no colony should form on the plates containing IPTG, 3e5 colonies formed on the plates containing IPTG might have resulted from the inherent heterogeneity of the cells within a population. 3.3. Screening system for orthogonal suppressor tRNAs in Ec Based on the above results, we designed a screening system for orthogonal suppressor tRNAs. The tRNA mutation library was constructed using the high-copy-number plasmid pMD18-T and named pMD-tRNAlib. Cognate aaRS was constructed in the vector pACYC184, which is compatible with pMD18-T and named pAC-RS. The plasmid pMD-tRNAlib expressed the suppressor tRNA, and pAC-RS expressed aaRS. They were then co-transformed into the Ec strain CSH108 and screened for suppressor tRNA toxicity combined with blueewhite selection (Fig. 4).
Fig. 3. Effect of the copy number of suppressor tRNA on different subtypes of E. coli. The plasmids pAC-tRNA and pMD-tRNA were transformed into the DH10B, CSH108, and DH5a respectively. The pACYC184 and pMD18-T were also transformed into the corresponding cells to afford the control group. Each bar represents the mean of OD600 of triplicate samples SD.
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Fig. 4. Screening system for orthogonal suppressor tRNA. The plasmid pMD-tRNAlib expressed the suppressor tRNA while pAC-RS expressed aaRS; they were then co-transformed into the E. coli strain CSH108. When a member of the suppressor tRNA library is aminoacylated by endogenous E. coli aaRS, a large number of transcripts of suppressor tRNA would lead to host cell death. Meanwhile, as the strain CSH108 contains an episomal lacZ gene with an amber nonsense codon, if a suppressor tRNA is aminoacylated by its cognate aaRS, full-length b-galactosidase would be produced, resulting in a blue colony on the plate. Cells harboring nonfunctional tRNAs would yield a white colony due to in-frame translation termination.
To verify whether the suppressor tRNA with poor orthogonality misaminoacylated in the host cells and could be eliminated by our screening system, pAC-tRNATyr-CCA and pMD-tRNATyr-CCA were transformed into CSH108-competent cells. The strain CSH108 contained an episomal lacZ gene with an amber nonsense codon. If the suppressor Mj tRNATyr could be aminoacylated by Ec aaRS, then a full-length b-galactosidase would be produced, resulting in a blue lawn on the plate containing X-gal. As shown in Fig. 5A, a blue lawn formed on the plate, indicating that Mj tRNATyr could be aminoacylated by Ec aaRS. Our results are in accordance with those reported in a previous paper [17]. By contrast, the cells containing pMD-tRNATyr-CCA did not grow on the LB plate, so no lawn formed (Fig. 5B). Thus, cells containing the suppressor Mj tRNATyr with the CCA terminus could be eliminated by our screening system. To verify if the blueewhite selection using CSH108 could separate the orthogonal suppressor tRNA and non-functional suppressor tRNA, the plasmid pMD-mu-tRNATyr was constructed by inserting the mu-tRNATyr gene into pMD18-T. The mu-tRNATyr gene is a suppressor tRNA mutant derived from Mj tRNATyr, which has better orthogonality based on previous reports [14]. Then, the pACRS expressing TyrRS was co-transformed with pMD-mu-tRNATyr into CSH108. The plasmid pMD18-T without the mu-tRNATyr gene was also co-transformed with pAC-RS into CHS108 as a control. The CSH108 cells containing pAC-RS and pMD-Mj mu-tRNATyr formed blue colonies on the LB plate (Fig. 5C), whereas the CSH108 cells containing pAC-RS and pMD18-T formed white colonies on the plate (Fig. 5D). Considering that orthogonal suppressor tRNAs are used mainly in gene code expansion, Mj TyrRS mutants that can aminoacylate suppressor tRNAs with unnatural amino acids (e.g., p-acetylphenylalanine and p-nitrophenylalanine) were also verified. The CSH108 cells containing pAC-AcRS, expressing the TyrRS mutant that could recognize p-acetylphenylalanine, and pMD-Mj mutRNATyr formed blue colonies (Fig. 5E). Similarly, the CSH108 cells containing pAC-NiRS, expressing the TyrRS mutant that could
recognize p-nitrophenylalanine, and pMD-Mj mu-tRNATyr also formed blue colonies on the LB plate (Fig. 5F). 3.4. Suppressor tRNA library design, construction, and screening To demonstrate the effectiveness of our design strategy, we constructed the suppressor tRNA library derived from Mj tRNATyr. Based on a previous report, 11 non-conserved nucleotides (C16, C17, U17a, U20, C32, G37, A38, U45, U47, A59, and U60) were randomized to maximize the likelihood of identifying a mutant tRNA with high orthogonality. The conserved nucleotides were not randomized to maintain the tertiary interactions that stabilize the L-shaped structure of the tRNA (Fig. 6A) [9]. The suppressor tRNA library was screened using the strategy described earlier. As expected, when co-transformed with the TyrRS mutant specific to p-nitrophenylalanine, colonies with different colors (white, light blue, or deep blue) formed on the LB plate containing p-nitrophenylalanine (Fig. 6B). The white colonies indicate that the suppressor tRNA mutant in that colony was nonfunctional. A deep blue color implies that a full-length b-galactosidase was produced in the host cell, indicating that the suppressor tRNA mutant in that colony could be more effectively aminoacylated by the aaRS mutant specific to p-nitrophenylalanine. Therefore, three deep blue colonies were selected and cultured in the LB medium. The sequencing results of the three colonies revealed that three independent genes for suppressor tRNAs were isolated (Fig. 6C). To confirm the properties of the selected suppressor tRNAs, another conventional assay based on the suppression of an amber codon in the CAT gene was conducted [8]. In the CAT assay, an amber codon was substituted for Asp112 in the CAT gene of pACYC184 to produce pAC-Cm. The genes encoding Mj TyrRS were inserted into the pre-digested pAC-Cm to construct the plasmid pAC-RS-Cm. The three selected suppressor tRNAs were separated and retransformed into Ec DH10B with pAC-Cm or pAC-RS-Cm. Then,
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Fig. 5. Blueewhite selection of CSH108 containing orthogonal suppressor tRNA and aaRS mutant (A) CSH108 cells containing pMD-tRNATyr-CCA; (B) CSH108 cells containing pACtRNATyr-CCA; (C) CSH108 cells containing pAC-RS and pMD-Mj mu-tRNATyr; (D) CSH108 containing pAC-RS and pMD18-T; (E) CSH108 cells containing pAC-AcRS, expressing the tyrosyl-tRNA synthetase mutant that could recognize p-acetylphenylalanine, and pMD-Mj mu-tRNATyr on the plate containing p-acetylphenylalanine; (F) CSH108 cells containing pAC-NiRS, expressing the tyrosyl-tRNA synthetase mutant that could recognize p-nitrophenylalanine, and pMD-Mj mu-tRNATyr on the plate containing p-nitrophenylalanine.
the DH10B cells were cultured in the LB medium containing various concentrations of chloramphenicol. Considering that the amber codon was introduced in the chloramphenicol-resistant gene in pAC-RS-Cm and pAC-Cm, the chloramphenicol tolerance of DH10B
depended on the aminoacylation of the tRNA mutant. As expected, the chloramphenicol tolerance of DH10B containing pMD-tRNA and pAC-RS-Cm reached 80 mg/mL, whereas that of DH10B containing pMD-tRNA and pAC-Cm was lower than 20 mg/mL (Fig. 7). These
Fig. 6. Screening results of the suppressor tRNA library (A) tRNA library derived from M. jannaschii tRNATyr. Randomly mutated nucleotides (N), each mutant clone in an independent culture, are shaded in black. (B) CSH108 cells containing pAC-NiRS, expressing the tyrosyl-tRNA synthetase mutant that could recognize p-nitrophenylalanine, and pMDtRNAlib on the plate containing p-nitrophenylalanine; (C) DNA sequences of mutant suppressor tRNAs selected from the suppressor tRNA library.
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Fig. 7. CAT assay results of selected suppressor tRNAs. Dark gray line represented the chloramphenicol tolerance of DH10B containing the pMD-tRNA which expresses the selected suppressor tRNA and pAC-RS-Cm which express the M. jannaschii tyrosyl tRNA synthetase. Light gray line represented the chloramphenicol tolerance of DH10B containing the pMD-tRNA and pAC-Cm that without the M. jannaschii tyrosyl tRNA synthetase gene.
results indicate that the selected suppressor tRNAs could be aminoacylated by Mj TyrRS but not by any Ec aaRS. Thus, the selected suppressor tRNAs were orthogonal based on the CAT assay. 4. Discussion The primary goal of our study is to establish a high-throughput screening approach for obtaining orthogonal suppressor tRNAs based on their species-specific toxicity. Therefore, we first verified if the species-specific toxicity of an amber suppressor tRNA toward the host depends on its orthogonality. The suppressor tRNATyr from Ec completely inhibited the growth of Ec cells under the conditions used, which may have resulted in the production of abnormal proteins in bacteria via readthrough of a stop codon by the suppressor tRNAs. Although the amber codon TAG is the least used nonsense triplet in Ec (w9%), 320 genes in Ec terminate in the UAG codon, including 44 essential genes [18]. By contrast, Mb tRNAPyl is
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a natural evolutionary suppressor tRNA that can be aminoacylated by pyrrolysyl-tRNA synthetase (PylRS). When transformed into Ec, Mb PylRS and tRNAPyl function as an orthogonal pair in vivo [19]. The special structure of tRNAPyl renders it incapable of aminoacylation by any Ec aaRS. Therefore, tRNAPyl is not toxic to the Ec host. In contrast to tRNAPyl, the suppressor Mj tRNATyr is a substrate for the endogenous Ec synthetases [15]. Our results also showed that cells harboring the suppressor Mj tRNATyr conduct a translational readthrough of the b-galactosidase gene containing the amber codon mutant. Hence, the poor orthogonality leads to a wild suppressor Mj tRNATyr that can be charged by the Ec endogenous aaRS and cause perturbation to the growth of the host cells. Similarly, Sc tRNATrp was proven to be a substrate for Ec lysyl tRNA synthetase [3], Therefore, the cells harboring Sc tRNATrp grew slower than the control group. The tRNA genes of both Mj tRNATyr and Sc tRNATrp lack the CCA sequence at their 30 end. These tRNAs need the addition of the CCA sequence in the maturation process by ATP (cytidine triphosphate):tRNA nucleotidyl transferase, which is present in archaeans, bacteria, and eukaryotes, as described by Woese [20]. When the complete CCA sequence is absent in the tRNA precursor, as in the cases of Mj tRNATyr and Sc tRNATrp, RNase BN removes the extra nucleotides from the 30 terminus of the precursor RNA and then the CCA 30 -OH terminus could be added by the CCA enzyme [21]. As a result, the tRNA without the CCA sequence at the 30 end requires more steps to mature before it can be aminoacylated, thereby inhibiting the growth of the host cells, but not being lethal to the host cells. A recent kinetic report has revealed that the Ec CCA enzyme has the innate ability to discriminate against tRNA backbone damage at each step of the CCA synthesis [22], leading to the inactivation of extraneous suppressor tRNA mutants. These results revealed that the suppressor tRNA gene with a CCA sequence at the 30 end presented greater toxicity to the host. Thus, the toxicity of suppressor tRNAs is a consequence of stop codon readthrough and is closely related to the orthogonality of tRNAs. A prerequisite to building a negative screening system is effectively eliminating tRNA mutants with poor orthogonality, which means that the toxicity of the suppressor tRNAs must be severe enough to kill the host. Our results revealed that the suppressor tRNAs inserted into high-copy-number plasmids exhibited stronger effects in inhibiting host growth. The readthrough event may have been a result of competition between release factors and suppressor tRNAs for the amber codon. Thus, increasing the copy numbers of suppressor tRNAs increases the suppression of all amber codons in the cell, enhancing the readthrough of stop codons on chromosomal genes [23]. To confirm further the effect of copy numbers on the host, an inducible copy number plasmid, pSB2K3, was used in this study. The pSB2K3 plasmid contains the P1 lytic replication origin, which is under the control of a lacI-regulated promoter. When induced with IPTG, P1 lytic replication proteins are produced. Thus, the P1 lytic replication origin is activated, raising the copy number to very high levels (http://partsregistry. org/Part:pSB2K3). The cells harboring pSB-tRNA formed less colonies on the plate containing IPTG, demonstrating that suppressor tRNAs in high-copy-number plasmids could effectively eliminate tRNA mutants with poor orthogonality. Therefore, this high-copynumber plasmid pMD18-T was selected as the screening vector. Another advantage of pMD18-T is its convenience for sequencing because it is derived from pUC18 and its sequencing primers are widely commercialized. Based on these results, we designed a screening system for orthogonal suppressor tRNAs. A suppressor tRNA mutant library was inserted into pMD18-T to form negative screening components. When a member of the suppressor tRNA library was aminoacylated by endogenous Ec aaRS (i.e., not orthogonal to Ec aaRS),
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many transcripts of suppressor tRNAs would lead to host cell death. Only cells harboring orthogonal tRNAs or non-functional tRNAs would survive. Furthermore, given that the strain CSH108 contained an episomal lacZ gene with an amber nonsense codon, a fulllength b-galactosidase was produced upon the aminoacylation of a suppressor tRNA by its cognate aaRS. Thus, a blue colony formed on the plate containing the galactoside analog X-gal. By contrast, the cells harboring non-functional tRNAs yielded a white colony because of the in-frame translation termination. In addition, the colonies presented various shades of blue, suggesting that the suppressor tRNA mutant in the deep blue colony may have been more effectively aminoacylated by its cognate aaRS than that in the light blue colony. Thus, by combining the analysis on the toxicity of the suppressor tRNAs toward the host with blueewhite detection, we developed a convenient screening system for identifying orthogonal suppressor tRNAs, which could serve as a general platform for generating tRNA/aaRS pairs. Acknowledgments This work has been supported by the Chinese National Natural Science Foundation (31200694 and 81273426), Important National Science & Technology Specific Projects (2009ZX09102-224), National Fund for Fostering Talents of Basic Science (J0630858) and Qing Lan Projects. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biochi.2012.12.010. References [1] Q. Wang, A.R. Parrish, L. Wang, Expanding the genetic code for biological studies, Chem. Biol. 16 (2009) 323e336. [2] J. Xie, P.G. Schultz, A chemical toolkit for proteins e an expanded genetic code, Nat. Rev. Mol. Cell. Biol. 7 (2006) 775e782. [3] R.A. Hughes, A.D. Ellington, Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA, Nucleic Acids Res. 38 (2010) 6813e6830. [4] J.M. Schrader, S.J. Chapman, O.C. Uhlenbeck, Understanding the sequence specificity of tRNA binding to elongation factor Tu using tRNA mutagenesis, J. Mol. Biol. 386 (2009) 1255e1264.
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