Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNAPyl Pairs in the Escherichia coli BL21(DE3) Cell Strain

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNAPyl pairs and cross recognition between nonsense codons and various tRNAPyl anticodons in the Escherichia coli BL21(DE3) cell strain are reported. is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, Nε-tert-butoxycarbonyl-l-lysine (BocK). Similar to, is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS- pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS- pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.


Introduction
Pyrrolysine (Pyl, Figure 1), the 22 nd proteinogenic amino acid that was originally discovered in methanogenic methylamine methyltransferase, is genetically encoded by the RNA nucleotide triplet UAG, a stop codon that halts translation of mRNA during a regular protein translation process [1]. The delivery of Pyl to ribosome is mediated by a unique tRNA, tRNA Pyl CUA that is specifically aminoacylated by a unique aminoacyl-tRNA synthetase (aaRS), pyrrolysyl-tRNA synthetase (PylRS) [2]. tRNA Pyl CUA contains a special nucleotide triplet CUA, an anticodon that recognizes a UAG stop codon in mRNA. Unlike tRNA Sec that needs a special elongation factor (SelB in E. coli and EFsec in mammalian cells) and an mRNA secondary structure for its binding to the ribosome A site and recognition of a UGA stop codon for the delivery of selenocysteine, tRNA Pyl CUA hijacks the regular translation elongation process to suppress a UAG codon for the incorporation of Pyl [3][4][5]. Previous studies also demonstrated that PylRS shows remarkably high substrate promiscuity and is able to charge tRNA Pyl CUA with a variety of noncanonical amino acids (NAAs). For these reasons and due to the naturally high orthogonality of the PylRS-tRNA Pyl CUA pair in bacteria, yeast, and mammalian cells, this pair has been directly transferred to E. coli, Saccharomyces cerevisiae, and human cells for the genetic incorporation of more than ten lysine derivatives including N e -tert-butoxycarbonyl-L-lysine (BocK) into proteins at amber mutation sites [6][7][8][9][10][11][12]. Engineered PylRS-tRNA Pyl CUA pairs have also been used to genetically encode other lysine derivatives and even phenylalanine derivatives that are structurally distinctive from Pyl [13][14][15][16][17][18][19][20][21]. The genetic incorporation of these NAAs into proteins and their following modifications allow a variety of biochemistry studies such as the functional investigation of protein posttranslational modifications, protein folding dynamic analysis, biosensor development, tracking signal transduction processes, and probing enzyme mechanisms [22]. Pioneered by Schultz and coworkers on the incorporation of two different NAAs [23], two methods were recently developed independently in the Chin Group and our group that couple the wild-type or a derivative PylRS-tRNA Pyl pair with another suppressing aaRS-tRNA pair for the genetic incorporation of two different NAAs into one protein in E. coli [24,25]. The method developed in the Chin Group used one amber codon and one quadruple AGGA codon that were suppressed by the PylRS-tRNA Pyl CUA pair and an evolved M. jannaschii tyrosyl-tRNA synthetase MjTyrRS-tRNA Tyr UCCU pair, respectively to code two different NAAs. A specially engineered ribosome, Ribo-Q1, was used to improve the AGGA suppression level. Our method relied on the suppression of two stop codons, namely one amber UAG codon and one ochre UAA codon, which was achieved by genetically encoding an evolved MjTyrRS-tRNA Tyr CUA pair for amber suppression and a wild type or evolved PylRS-tRNA Pyl UUA pair for ochre suppression in E. coli. The genetic incorporation of two different NAAs into one protein can be potentially applied to install a FRET pair in a protein for conformation and dynamic studies as we demonstrated in a separate publication [26], synthesize proteins with two different posttranslational modifications for their functional investigation, and build phage-displayed peptide libraries with the expanded chemical diversities.
Although the PylRS-tRNA Pyl CUA pair has been used extensively for the genetic incorporation of different NAAs in the past few years, two questions related to the pair have not been fully addressed. While our lab and other groups have showed that mutating the anticodon of tRNA Pyl CUA does not significantly affect its interaction with the catalytic domain of PylRS [27], further investigation need to be done to determine whether we can directly use mutant tRNA Pyl forms for the genetic incorporation of NAAs at an opal or ochre codon or even a sense codon in E. coli. Another study is necessary to clarify whether an aminoacylated tRNA Pyl UUA can lead to amber suppression since a UUA anticodon can recognize a UAG codon based on the wobble hypothesis [28]. In this study, we attempted to address these two questions and carried out all the experiments in the E. coli BL21(DE3) cell strain which has been broadly used for the genetic incorporation of NAAs.

Materials
Phusion high-fidelity DNA polymerase, T4 DNA ligase, T4 polynucleotide kinase, and restriction enzymes were purchased from New England Biolabs. Oligonucleotide primers were ordered from Integrated DNA Technologies. Ni-NTA superflow resins were purchased from Qiagen. All polymerase chain reactions (PCRs) were performed using Phusion high-fidelity DNA polymerase. BocK was purchased from Chem Impex. p-Azido-Lphenylalanine (AzF) was synthesized according to a revised literature procedure [29].

Plasmids
Plasmid pETtrio-pylT(UUA)-PylRS-MCS was derived from pPylRS-pylT-GFP1TAG149TAA [25] and carries a tRNA Pyl UUA gene (a C34U mutant form of tRNA Pyl CUA ) under control of the lpp promoter and the rrnC terminator, the wild type Methanosarcina mazei PylRS gene under control of the glnS promoter and terminator, and multiple cloning sites including NcoI, NotI, SalI and KpnI targeted sites under control of the T7 promoter and terminator. Plasmid pETtrio-pylT(UUA)-PylRS-sfGFP134TAG that carries an additional superfolder green fluorescent protein (sfGFP) gene with an amber mutation at N134 (sfGFP134TAG) was constructed by cloning the sfGFP134TAG gene to the NcoI and KpnI sites of pETtrio-pylT(UUA)-PylRS-MCS.

Amber, Opal, and Ochre Suppression
Plasmids pETtrio-pylT(CUA)-PylRS-sfGFP134TAG, pETtrio-pylT(UCA)-PylRS-sfGFP134TGA, and pETtrio-pylT(UUA)-PylRS-sfGFP134TAA were individually used to transform E. coli BL21(DE3) cells. For each plasmid, a single colony was selected and allowed to grow in 5 mL of LB medium with 100 mg/mL ampicillin at 37uC overnight. The overnight culture was inoculated into 200 mL of 2YT medium with 100 mg/mL ampicillin and allowed to grow at 37uC to OD 600 ,1.2. 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and 5 mM BocK were then added to the medium to induce expression of sfGFP. Control experiments in which only 1 mM IPTG was added to the medium were also carried out. The induced cells were allowed to grow at 37uC overnight and then collected by centrifugation (4,200 rpm for 20 min). The collected cells were resuspended in 35 mL of lysis buffer (50 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed by sonication in an ice water bath. The lysed cells were clarified by centrifugation (10,000 rpm for 1 h). The supernatant was decanted and let bind to 5 mL of Ni-NTA superflow resins at 4uC for 1 h. The mixture of the supernatant and resins was then loaded to an empty Qiagen Ni-NTA superflow cartridge. The resins were washed with 5 volume times of lysis buffer and sfGFP was then eluted with buffer (50 mM HEPES, 300 mM NaCl, 250 mM imidazole, pH 8.0). To further purify the expressed sfGFP, the protein was equilibrated against buffer A (20 mM Bis-Tris, pH 6.1) and then loaded to a monoS column from GE Health Science. The protein was eluted out by running a gradient from buffer A to 100% of buffer B (20 mM Bis-Tris, 1 mM NaCl, pH 6.1). The finally purified protein was then concentrated to a desired volume and analyzed by SDS-PAGE. To analyze the purified protein by electrospray ionization mass spectrometry (ESI-MS) analysis, the buffer of the purified protein was changed to the phosphate buffer saline. Without further indication, protein purification and characterization in the following experiments were the same.

Basal Suppression at an Opal UGA Codon
Plasmid pETtrio-sfGFP134TGA was used to transform E. coli BL21(DE3) cells. A single colony was then selected to do protein expression that was induced by the addition of 1 mM IPTG.

Competitive Recognition of the Third Nucleotide of an Amber Codon
Plasmid pEVOL-AzFRS was a gift from Dr. Peter Schultz at Scripps Research Institute [30]. It carries one tRNA Tyr CUA gene under control of a proK promoter and a proK terminator, an evolved AzF-specific MjTyrRS (AzFRS) gene under control of a glnS promoter and a glnS terminator, and an AzFRS gene under control of a pBAD promoter. This plasmid together with pETtrio-pylT(UUA)-PylRS-sfGFP134TAG was used to co-transform E. coli BL21(DE3) cells. One single colony was selected and allowed to grow in 5 mL of LB medium with 100 mg/mL ampicillin and 34 mg/mL chloramphenicol at 37uC overnight. This overnight culture was inoculated into 200 mL of 2YT medium with 100 mg/ mL ampicillin and 34 mg/mL chloramphenicol and allowed to grow to OD 600 ,1.2. Expression of sfGFP was then induced. Four induction conditions were tested, including (1) 1 mM IPTG only, (2) 1 mM IPTG and 1 mM AzF, (3) 1 mM IPTG and 5 mM BocK, and (4) 1 mM IPTG, 1 mM AzF, and 5 mM BocK.

AGG Codon Suppression
Plasmid pETtrio-pylT(CCU)-PylRS-sfGFP2AGG was used to transform E. coli BL21(DE3) cells. A single colony was then selected and allowed to grow in 5 mL of LB medium with 100 mg/ mL ampicillin at 37uC overnight. This overnight culture was then inoculated into 500 mL of 2YT medium with 100 mg/mL ampicillin and allowed to grow to OD 600 ,1.2. Two conditions were used to induce sfGFP expression. One is the addition of 1 mM IPTG and the other is the addition of 1 mM IPTG and 5 mM BocK.

Amber, Opal, and Ochre Suppression Efficiencies of the PylRS-tRNA Pyl Pairs
To demonstrate amber suppression efficiency of the PylRS-tRNA Pyl CUA pair, E. coli BL21(DE3) cells transformed with pETtrio-pylT(CUA)-PylRS-sfGFP134TAG were used to express sfGFP both in the absence and in the presence of 5 mM BocK, a substrate of PylRS. Without BocK in the growth medium, low level of sfGFP expression was observed. On the contrary, the addition of BocK promoted sfGFP overexpression ( Figure 2A). The ESI-MS analysis of the purified sfGFP displayed two major mass peaks at 27,81061 Da and 27,94161Da that agree well with the theoretical molecular weights of sfGFP with BocK incorporated at N134 (27,940 Da for the full-length protein; 27809 Da for the full-length protein without the first methionine (M1)) ( Figure 2B1). E. coli BL21(DE3) cells transformed with pETtrio-pylT(UUA)-PylRS-sfGFP134TAA showed an undetectable expression level of sfGFP when BocK was absent in the growth medium. The addition of BocK induced sfGFP expression ( Figure 2A). The ESI-MS analysis of the purified sfGFP showed two mass peaks (27,94061 Da and 27,80961 Da) that agree well with the theoretical molecular weights of sfGFP with BocK incorporated at N134 ( Figure 2B2). In comparison to amber suppression, the expression level of sfGFP using ochre suppression is lower. E. coli BL21(DE3) cells transformed with pETtrio-pylT(UCA)-PylRS-sfGFP134TGA exhibited a high expression level of sfGFP both in the absence and in the presence of BocK in the growth medium ( Figure 2A). The ESI-MS analysis of purified sfGFP expressed in the absence of BocK showed a mass peak at 27,89961 Da that clearly matched a Trp residue at N134 of sfGFP (calculated mass: 27,898 Da) ( Figure 2B3). The ESI-MS analysis of sfGFP expressed in the presence of BocK displayed a very interesting spectrum. Mass peaks for both Trp residue at N134 of sfGFP (27,90061 Da) and BocK residue at N134 of sfGFP (27,94161 Da) were observed. The mass peak for the Trp isoform was much more intensive than the BocK isoform ( Figure 2B4).
To test whether the incorporation of Trp at the 134 position is related to the nucleotide contents of mRNA around the UGA codon, plasmid pETtrio-pylT(UCA)-PylRS-sfGFP2TGA was constructed to test the opal suppression. E. coli BL21(DE3) cells transformed with pETtrio-pylT(UCA)-PylRS-sfGFP2TGA displayed high sfGFP expression levels both in the absence and in the presence of BocK that were much higher than in cells transformed with plasmid pETtrio-pylT(UCA)-PylRS-sfGFP134TGA and grown in the same conditions ( Figure 3A).    The suppression efficiencies of three mutated forms of tRNA Pyl UCA with mutations as G73A, G73C, and G73U, respectively were also examined. Cells transformed with either pETtrio-pylT(UCA)G73A-PylRS-sfGFP134TGA or pETtrio-pylT(U-CA)G73C-PylRS-sfGFP134TGA displayed similar expression levels of sfGFP both in the presence and in the absence of BocK( Figure 5A). However, cells transformed with pETtrio-pylT(UCA)G73U-PylRS-sfGFP134TGA showed significantly different expression levels of sfGFP when grown in the absence and in the presence of BocK. The ESI-MS analysis of sfGFP expressed in the absence of BocK showed a major mass peak at 27,89561 Da that matches sfGFP with Trp incorporated at N134 ( Figure 5B1). The addition of 5 mM BocK promoted the sfGFP expression level to increase. The ESI-MS analysis of the purified protein confirmed sfGFP with BocK incorporated at N134 became dominant ( Figure 5B2). The intensity of the mass peak at 27,94061 Da that matches sfGFP with BocK incorporated at N134 is roughly twice of the mass peak at 27,89861 Da that matches sfGFP with Trp incorporated at N134.

Anticodon-codon Cross Recognition
Transforming E. coli BL21(DE3) cells with pETtrio-pylT(CUA)-PylRS-sfGFP134TAA followed by growing cells in the presence of 5 mM BocK did not lead to detectable expression of sfGFP. E. coli BL21(DE3) cells transformed with pETtrio-pylT(UUA)-PylRS-sfGFP134TAG showed a detectable but low level of sfGFP expression (less than 1 mg/L) ( Figure 6). To see whether BocK promoted suppression at the amber mutation site, E. coli BL21(DE3) cells transformed with pETtrio-pylT(UUA)-PylRS-sfGFP134TAG was also grown in the absence of BocK. As shown in Figure 7A, the sfGFP expression levels both in the absence and in the presence of BocK were very similar. Addition of BocK did not lead to significant increase of amber suppression. As shown in Figures 7B1&B2, sfGFP expressed in both conditions displayed mass peaks (27,83961 Da and 27,71061 Da in Figure 7B1 and 27,84061 Da and 27,70961 Da in Figure 7B2) that match sfGFP with glutamic acid (Glu), lysine (Lys), or glutamine(Gln) incorporated at N134. Figure 7B2 did show a mass peak at 27,93961 Da that matches the molecular weight of sfGFP with BocK incorporated at N134. However, its intensity was much lower than the mass peak of sfGFP at ,27,84061 Da.
Experiments were also carried out to demonstrate that the PylRS-tRNA Pyl UUA pair does not interfere with suppression of an amber mutation mediated by an evolved MjTyrRS-tRNA Tyr CUA pair. Two plasmids, pETtrio-pylT(UUA)-pylRS-sfGFP134TAG and pEVOL-AzFRS were used to transform E. coli BL21(DE3)cells. As shown in Figure 8A, growing the transformed cells in four conditions led to different expression levels of sfGFP. When no NAA or only 5 mM BocK was provided in the medium, only a detectable but very low level of sfGFP expression (less than 1 mg/ L) was detected. However, addition of 1 mM AzF to the medium supplemented with or without BocK promoted sfGFP overexpression. The ESI-MS analysis of purified sfGFP in all four conditions  Figure 8B4) matches the calculated molecular weight (27,901 Da) of sfGFP with AzF incorporated at N134, a mass peak for sfGFP expressed in two conditions without the supplement of AzF (27,85961 Da in Figure 8B1 and 27,86061 Da in Figure 8B3) does not match the molecular weight of sfGFP with either Lys/ Glu/Gln or BocK incorporated at N134. Instead, this mass peak agrees well with the molecular weight of sfGFP with Phe incorporated at N134 (calculated mass: 27,859 Da).

AGG Codon Suppression
E. coli BL21(DE3) cells transformed with pETtrio-pylT(CCU)-PylRS-sfGFP2AGG showed similar sfGFP expression levels both in the absence and in the presence of BocK ( Figure 9A). sfGFP proteins expressed in both conditions displayed one major mass peak at 27,895 Da that agrees well with the theoretical molecular weight of sfGFP with arginine (Arg) incorporated at N134 (calculated mass: 27,896 Da for the full-length protein) ( Figure 9B).

Discussion
Basal Nonsense Suppression in the E. coli BL21(DE3) Cell Strain E. coli BL21(DE3) cells transformed with pETtrio-pylT(CUA)-PylRS-sfGFP134TAG and grown in the absence of BocK yielded a sfGFP expression level close to 1 mg/L. The ESI-MS spectrum of the purified sfGFP clearly indicated a Lys, Glu, or Gln residue at the amber mutation site. Since PylRS does not recognize Lys, Glu, and Gln and tRNA Pyl CUA itself does not mediate detectable amber suppression in the E. coli Top10 cell strain (data not shown), this low but detectable sfGFP expression level was due to the basal amber suppression in the E. coli BL21(DE3) cell strain [6,27]. This basal amber suppression that was also demonstrated in one separate study from us [27] arises possibly from the recognitions of the UAG codon by its near-cognate tRNAs including tRNA Lys UUU / tRNA Glu UUC /tRNA G ln CUG [31,32]. A similar test with E. coli BL21(DE3) cells transformed with pETtrio-pylT(UCA)-PylRS-sfGFP134TGA and grown in the absence of BocK yielded a sfGFP expression level of 8 mg/L. The ESI-MS spectrum of the purified protein showed a Trp residue at N134 of sfGFP. To rule out the possibility that tRNA Pyl UCA was charged by E. coli tryptophanyl-tRNA synthetase  . This high efficiency to read across an opal codon with the binding of a near-cognate tRNA Trp may correlate with the short distance from the opal codon to the start codon. In contrary to UAG and UGA codons, the UAA codon displays high translation termination stringency in the E. coli BL21(DE3) cell strain. Cells transformed with pETtrio-pylT(CUA)-PylRS-sfGFP134TAA showed an undetectable basal ochre suppression level. This can be explained from several aspects. UAG and UGA are recognized by release factor 1 and release factor 2, respectively, whereas UAA is recognized by both release factor 1 and release factor 2. Its recognition by both release factor proteins, in theory, makes the translation termination at UAA more stringent than the other two stop codons. Another reason lies at the nucleotide contents of UAA. Unlike UAG and UGA that  could involve a GC base pair interaction, UAA could only form AU pairs or wobble pairs. Its interactions with tRNAs are relatively weak, making its misrecognition less possible than UAG and UGA.

Amber, Opal, and Ochre Suppression Efficiencies of the PylRS-tRNA Pyl Pairs
When co-expressed with PylRS, all three tRNA Pyl isoforms tRNA Pyl CUA ,tRNA Pyl UCA , and tRNA Pyl UUA are capable to deliver BocK at their corresponding codon sites. tRNA Pyl CUA is orthogonal in E. coli and displays the highest efficiency in all three isoforms. Given that E. coli BL21(DE3) cells transformed with pETtrio-pylT(UUA)-PylRS-sfGFP134TAA did not show a detectable expression level of sfGFP in the absence of BocK, we could conclude that tRNA Pyl UUA is fully orthogonal in E. coli. In comparison to sfGFP expressed in cells transformed with pETtrio-pylT(CUA)-PylRS-sfGFP134TAG and grown in the presence of BocK, the sfGFP expression level in cells transformed with pETtrio-pylT(UUA)-PylRS-sfGFP134TAA and grown in the presence of BocK is five times lower. The low ability of tRNA Pyl UUA to deliver BocK is possibly due to the relative weak base pair interactions between its UUA anticodon, its UAA stop codon and the availability of both release factor 1 and release factor 2 to stop the translation at a UAA stop codon. In any case, the ochre suppression level achieved by the PylRS-tRNA Pyl UUA pair is sufficient to promote overexpression of a protein with an ochre mutation. Although a BocKaminoacylated tRNA Pyl UCA is able to suppress an opal codon for the incorporation of BocK, it does not inhibit the high incorporation of Trp at the same site. As an exogenous tRNA, the sequence and structure of tRNA Pyl UCA may not be optimal for the protein translation process in E. coli. When facing a competition from E. coli tRNA Trp , the recognition of tRNA Pyl UCA by the E. coli translation machinery may be inhibited. Since the 73rd nucleotide serves as a strong recognition element for most tRNAs [33], it was mutated in tRNA Pyl UCA to U, A, and C and searched for a mutant that show a higher opal suppression efficiency. When co-expressed with PylRS in the presence of BocK, tRNA Pyl UCA (G73U)led to higher incorporation level of BocK compared to Trp at the same opal mutation site. However, further mutagenesis with tRNA Pyl UCA (G73U)is necessary to fully inhibit the incorporation of Trp at an opal mutation site.

Anticodon-codon Cross Recognition
The wobble hypothesis was first introduced by Francis Crick in 1966 to explain the observation that a single tRNA is able to efficiently recognize multiple codons [28]. Based on this hypothesis, an ochre suppressor tRNA UUA is also capable of recognizing an amber UAG codon. This is a concern when both UAG and UAA codons are used to code two different NAAs. However, cells transformed with pETtrio-pylT(UUA)-PylRS-sfGFP134TAG and grown in the presence of 5 mM BocK showed a sfGFP expression level close to that from the basal amber suppression. This suggests very weak recognition of UAG bytRNA Pyl UUA . Weak base pairing interactions involved with the UUA anticodon may contribute to the weak recognition of UAG. However, this is certainly not the determining factor since other tRNAs such as tRNA Lys UUU is also involve weak base pairing interactions to recognize multiple codons. One possible explanation for this weak recognition of UAG by tRNA Pyl UUA is the tRNA modifications. All cognate tRNAs are known to exhibit similar affinities for the ribosome A site when they bind to corresponding codons [34,35]. This uniform binding is unexpected as certain codon-anticodon interactions are expected to be more stable than others due to factors such as the GC base pair content. It has been proposed that the specific sequence and post-transcriptional modification status of the tRNA in the region near the anticodon is tuned to ensure nearly indistinguishable binding of tRNAs to the ribosome A site [36][37][38]. This has been the case for tRNAs such as tRNA Lys UUU in which both nucleotides at 34 and 37 are post-transcriptionally modified to achieve similar recognitions of AAA and AAG codons [38]. Unlike endogenous tRNAs that have corresponding modification enzymes, tRNA Pyl UUA is exogenous and may not be targeted by tRNA modification enzymes in E. coli. tRNA Pyl UUA without modifications at its anticodon loop likely has a very weak binding affinity to the ribosome A site to associate UAG. Our finding points out that wobble base pairing at the 39 nucleotide of a codon is not sufficient for recruiting a tRNA to the ribosome A-site. Additional interactions are required. This aspect needs to be further investigated. Since tRNA Pyl UUA has a low ability to recognize UAG, it is feasible to use an amber suppressor aaRS-tRNA pair and a wild type or evolved PylRS-tRNA Pyl UUA pair to code two different NAAs at amber and ochre mutation sites, respectively, in E. coli.
During the anticodon-codon cross recognition analysis to examine whether tRNA Pyl UUA can compete against tRNA Tyr CUA to bind UAG in the ribosome A site, we noticed that Phe was incorporated at N134 of sfGFP which was expressed in cells transformed with pEVOL-AzFRS and pETtrio-pylT(UUA)-PylRS-sfGFP134TAG and grown either in the absence or in the presence of BocK. We think AzFRS can recognize Phe, leading to the misincorporation of Phe. Since AzF is a hydrophobic amino acid and structurally very similar to Phe, one would expect that AzFRS that was originally evolved from MjTyrRS would probably recognize Phe at a relatively low level and charge tRNA Tyr CUA with Phe when AzF is absent in the medium. Although not clearly addressed in existing literature, most evolved MjTyrRS-tRNA Tyr CUA pairs did show significant background amber suppression even in minimal media [39,40]. Since most evolved MjTyrRS variants are for Phe derivatives, it is highly possible that background amber suppression caused by these evolved MjTyrRS-tRNA Tyr CUA pairs was due to their recognition of either Phe or Tyr or both. In this study, the background amber suppression induced by the AzFRS-tRNA Tyr CUA pair inhibited both the basal amber suppression level and amber suppression induced by the PylRS-tRNA Pyl UUA pair. This test also provided evidence that amber suppression mediated by the PylRS-tRNA Pyl UUA pair is too low to be a concern. It also points out that substrate specificities of evolved NAA-specific aaRSs need to be further characterized.

AGG Codon Suppression
Our lab and other groups have shown that mutating the anticodon of tRNA Pyl does not significantly affect its interactions with PylRS. Three tRNA Pyl isoforms that are specific for three stop codons are capable to deliver BocK at their corresponding codon sites when co-expressed with PylRS. We were also curious about the ability a tRNA Pyl isoform to suppress a sensing codon. We chose to test on the suppression of the AGG codon since it is a rarely used codon and tRNA Arg CCU is limitedly expressed in the E. coli BL21(DE3) cell strain [41]. However, E. coli transformed with pETtrio-pylT(CCU)-PylRS-sfGFP2AGG only expressed sfGFP with Arg at its S2 position in the presence of 5 mM BocK.
Increasing the BocK concentration to 10 mM did drive the expression of sfGFP with BocK incorporated at S2 of sfGFP, which was confirmed by the ESI-MS analysis of the purified proteins (data not shown). However, in comparison to the Argcontaining sfGFP isoform that showed a high intensity in the ESI-MS spectrum of the purified sfGFP, the intensity for the BocKcontaining sfGFP isoform was very low. This indicates tRNA Pyl CCU is charged by PylRS with BocK in E. coli and is able to deliver BocK at an AGG codon site. However, the PylRS-tRNA Pyl CCU pair cannot compete efficiently against the endogenous Arg incorporation system at the AGG codon. Similar to tRNA Pyl UCA , the sequence and structure of tRNA Pyl CCU may not be optimized for the protein translation machinery in E. coli.
In summary, the suppression efficiencies of the original and three tRNA Pyl variant, the cross recognition between nonsense codons and tRNA Pyl anticodons in the E. coli BL21(DE3) cell strain have been investigated. Among all tRNA Pyl isoforms, tRNA Pyl CUA has the highest suppression efficiency for the delivery of BocK at its corresponding codon. Besides its orthogonal nature in E. coli, tRNA Pyl UUA does not induce a significant level of suppression at an amber codon. This is contrary to the wobble hypothesis and makes it feasible to use amber suppressing aaRS-tRNA pair and the PylRS-tRNA Pyl UUA pair to code two different NAAs at amber and ochre codons respectively in E. coli. Our study also demonstrates the PylRS-tRNA Pyl CCU pair cannot efficiently deliver BocK at an AGG codon site. Further work to optimize the sequence and structure of tRNA Pyl CCU for the E. coli translation machinery may be necessary to increase the BocK incorporation efficiency and suppress the Arg incorporation at the AGG codon.