Efficient expression of orthogonal tRNA in mammalian cells
An efficient way of generating an orthogonal tRNA-synthetase pair is to import a tRNA-synthetase pair from species in a different kingdom
18,19, because the cross-aminoacylation between different species is often low. The tRNA
Tyr-TyrRS pair from the archaebacterium
Methanococcus jannaschii was successfully used in
E. coli. Similarly, the
E. coli. tRNA
Tyr-TyrRs pair has been used in yeast, and should be orthogonal in mammalian cells. To test and use
E. coli tRNAs, one challenge lies in the expression of functional
E. coli tRNAs in mammalian cells.
E. coli and mammalian cells differ significantly in tRNA transcription and processing.
E. coli tRNAs are transcribed by the sole RNA polymerase through promoters upstream of the tRNA structural gene. However, the transcription of mammalian tRNA genes depends principally on promoter elements within the tRNA known as the A and B box sequences, which are recognized by RNA polymerase III (pol III) and its associated factors
20. Whereas all
E. coli tRNA genes encode full tRNA sequences, mammalian tRNAs have the 3′-CCA sequence added enzymatically by the tRNA nucleotidyltransferase after transcription. In addition, the 5′- and 3′-flanking sequences, the removal of introns, and the export from nucleus to cytoplasm also affect mammalian tRNA expression and function. Owing to these differences,
E. coli tRNAs, especially those that diverge from the preserved eukaryotic A and B box sequences, are not efficiently biosynthesized or correctly processed in mammalian cells.
We reasoned that a pol III promoter lacking any requirement for intragenic elements could efficiently transcribe tRNAs without the preserved internal A and B boxes in mammalian cells. The H1 promoter
21 was chosen for the following reasons: (i) it drives the expression of human
H1RNA and thus is of mammalian origin; (ii) it is a type-3 pol III promoter that has no downstream transcriptional elements; (iii) the transcription initiation site of the H1 promoter is well defined, and can be used to generate the 5′ end of the tRNA without further post-transcriptional processing; and (iv) the H1 promoter has been successfully used to express short interfering RNAs in mammalian cells.
We developed a fluorescence-based functional assay in mammalian cells to identify those expression elements that can efficiently drive the transcription of
E. coli tRNAs to generate functional tRNAs in mammalian cells (). The gene for the candidate
E. coli amber suppressor tRNA (
, whose anticodon is changed to CUA to decode the amber stop codon TAG) is co-expressed with its cognate synthetase (aaRS). A TAG stop codon is introduced at a permissive site of the
GFP gene, and this mutant
GFP gene is co-expressed with the
-aaRS pair in mammalian cells. If the
is expressed and correctly processed to a functional tRNA, the synthetase will aminoacylate this tRNA with the cognate amino acid. The acylated
will suppress the TAG codon in the
GFP gene, producing full-length GFP and rendering cells fluorescent. By comparing the fluorescence intensities of cells, this method can also serve as a sensitive
in vivo assay for the orthogonality of the
to endogenous synthetases of host cells when the cognate
E. coli synthetase is not expressed, and for the activity of the orthogonal
towards an unnatural-amino-acid-specific mutant synthetase when the mutant synthetase is expressed in place of the cognate synthetase.
The
E. coli tyrosyl amber suppressor tRNA (
) was chosen as the candidate orthogonal tRNA because it is orthogonal to yeast synthetases and suppresses the amber stop codon efficiently in yeast when coexpressed with
E. coli TyrRS
22.
In vitro aminoacylation assays indicate that
E. coli TyrRS does not charge eukaryotic tRNAs
23. Mammalian and yeast cells are eukaryotic, so we reasoned that the
-
E. coli TyrRS pair should be orthogonal in mammalian cells. For 3′-end processing of the
, we used the 3′-flanking sequence of the human tRNA
fMet. The 5′- and 3′-flanking sequences of the human tRNA
fMet can drive the functional expression of
E. coli
(which has the A box and B box) in mammalian cells
24. To test the importance of the 3′-CCA trinucleotide, these nucleotides were included or removed in the tRNA gene, resulting in four expression cassettes (tRNA-1 to tRNA-4; ). For comparison, we made a control plasmid (tRNA-5) in which the
was placed downstream of the 5′-flanking sequence of the human tRNA
Tyr.
To compare the ability of different expression cassettes to generate functional tRNAs, we established a clonal stable HeLa cell line expressing the
GFP gene with a TAG stop codon introduced at the permissive site 182 (GFP-TAG HeLa). The tRNA-aaRS expression plasmid was transfected into the stable GFP-TAG HeLa cell line, and cells were analyzed with flow cytometry after 48 h. The total fluorescence intensity of the green fluorescent cells indicates the amount of GFP produced (). When
-TyrRS was not expressed, the fluorescence intensity of the GFP-TAG HeLa cell line was similar to that of HeLa cells, indicating that the background readthrough of the TAG codon in GFP is negligible. Using the 5′-flanking sequence of human tRNA
Tyr in tRNA-5, only weak amber suppression was detected, confirming that bacterial tRNAs without the preserved A and B boxes could not be functionally expressed in mammalian cells. The highest fluorescence intensity was found in cells transfected with tRNA-4, which was 71-fold higher than that of tRNA-5, indicating that the H1 promoter can drive the functional biosynthesis of
much more efficiently than the 5′-flanking sequence of the human tRNA
Tyr. This also indicates that the H1 promoter can generate the correct 5′-end of the tRNA directly from the transcription initiation site without the post-transcriptional processing that is necessary for endogenously expressed tRNAs. The fluorescence intensity of cells transfected with tRNA-2 was 10% of that of cells transfected with tRNA-4, indicating that the 3′-flanking sequence of the human tRNA
fMet is also needed for the efficient expression of the
. It is intriguing to find that functional tRNA was produced in mammalian cells transfected with tRNA-1 (21% of tRNA-4), in which the CCA trinucleotide but no 3′-flanking sequence is included, as mammalian cells do not encode the CCA in the tRNA gene. However, when both the CCA trinucleotide and the 3′-flanking sequence were included in tRNA-3, the fluorescence intensity dropped markedly to 1.3%.
We used Northern blotting to measure the transcription levels of the
expressed by different constructs in GFP-TAG HeLa cells (). Very low levels of
could be detected using an
-specific probe in samples transfected with tRNA-5, tRNA-3 or tRNA-2. By contrast, in cells transfected with tRNA-4 and tRNA-1, the amounts of
were about 93-fold and 19-fold higher, respectively, than in cells transfected with tRNA-5. The northern blotting data confirmed that the
was transcribed in HeLa cells, and the increase in tRNA transcription is consistent with the increase in fluorescence intensity measured by cytometry in different samples.
To test the orthogonality of the
to endogenous synthetases in HeLa cells, we removed the
E. coli TyrRS in tRNA-4 to express the
only. Transfection of the resultant plasmid into the GFP-TAG HeLa cell line did not change the fluorescence intensity of the cells, showing that the
is not aminoacylated by any synthetases in HeLa cells.
To test whether the H1 promoter, together with the 3′-flanking sequence, can be used to express other
E. coli tRNAs, we replaced the
in construct tRNA-4 with the
E. coli leucyl amber suppressor tRNA (
) and the TyrRS with the cognate leucyl-tRNA synthetase (LeuRS). When only the
was expressed, we saw no fluorescence change in the GFP-TAG HeLa cells, indicating that the
is also orthogonal in HeLa cells. By contrast, when the
-LeuRS pair was expressed, the GFP-TAG HeLa cells became very bright. The total fluorescence intensity was 104% of that of cells transfected with the
-TyrRS pair. The
does not have a fully matched A box, whereas the
has no fully matched A or B box sequences. Taken together, these results show that the H1 promoter can efficiently drive the expression of
E. coli tRNAs, regardless of the internal promoter elements, in mammalian cells, and that the transcribed tRNAs are functional for amber suppression.
Unnatural-amino-acid-specific aaRS for mammalian cells
Synthetases that are specific for a variety of unnatural amino acids have been evolved in
E. coli and later in yeast from large mutant synthetase libraries consisting of >10
9 members
7. Similar strategies cannot be practically employed in mammalian cells and neurons because the transfection efficiencies of these cells are lower by several orders of magnitude than those of
E. coli and yeast. As both yeast and mammalian cells are of eukaryotic origin, we decided to transfer the mutant synthetases that had been evolved in yeast to mammalian cells. To test the feasibility of this approach, the
E. coli TyrRS gene in the tRNA-aaRS expression plasmid () was replaced with the gene for OmeTyrRS, a synthetase specific for the unnatural amino acid
o-methyl-
L-tyrosine (OmeTyr; ). The resultant plasmid was transfected into the GFP-TAG HeLa cell line, and cells were grown in the presence and absence of OmeTyr. As shown in , in the absence of OmeTyr, these cells were virtually nonfluorescent and similar to the GFP-TAG HeLa cells, indicating that the expression of the
-OmeTyrRS pair does not suppress amber codons efficiently. When OmeTyr was added, 71% of cells (normalized to the total number of fluorescent cells transfected with the
and wild-type TyrRS) became fluorescent, indicating that OmeTyr was incorporated into the GFP. The incorporation efficiency is about 41% by comparing the total fluorescence intensity of these cells to the intensity of cells transfected with the
-TyrRS pair.
To determine whether the transfer strategy could be generally applied to other synthetases evolved in yeast, we next tested the BpaRS, a synthetase that is specific for
p-benzoylphenylalanine (Bpa; ). As expected, when the BpaRS was coexpressed with the
in the GFP-TAG HeLa cell line, 47% of cells were fluorescent in the presence of Bpa, and virtually no fluorescent cells (≤4%) were detected in the absence of Bpa. The incorporation efficiency of this unnatural amino acid is about 13% ().
In addition to tRNA-aaRS pairs derived from the
E. coli tRNA
Tyr-TyrRS, we also tested a tRNA-aaRS pair derived from
E. coli tRNA
Leu-LeuRS. The
and a mutant synthetase specific for a fluorescent unnatural amino acid 2-amino-3-(5-(dimethylamino)-naphthalene-1-sulfonamido)propanoic acid (DanAla)
25 () were expressed in the GFP-TAG HeLa cell line. DanAla was incorporated with 13% efficiency, and 42% of the cells became fluorescent ().
These results confirm that unnatural-amino-acid-specific synthetases evolved in yeast can generally be transferred for use in mammalian cells. The tested unnatural amino acids were incorporated less efficiently than Tyr by the wild-type TyrRS, indicating that the activities of the evolved synthetases are not optimal. More rounds of directed evolution of these synthetases in yeast might further improve their incorporation efficiencies.
Genetically encoding unnatural amino acids in neurons
We first investigated whether the H1 promoter and the 3′-flanking sequence identified in HeLa cells could also generate functional amber suppressor tRNAs in neurons. Mouse hippocampal neurons were transfected with two plasmids simultaneously (): the reporter plasmid
pCLHF-
GFP-
TAG encoding a mutant GFP (182TAG), and the expression plasmid encoding the
E. coli TyrRS, the
driven by either the H1 promoter or the 5′ flanking sequence of human tRNA
Tyr, and a red fluorescent protein, mCherry, as an internal marker for transfection. Fluorescence microscopy was used to look for red transfected cells, and then to image their green fluorescence. The presence of green fluorescence in transfected cells indicates that functional
is biosynthesized to incorporate Tyr at the 182TAG position of the
GFP gene. As shown in , neurons transfected with the expression plasmid in which the
was driven by the H1 promoter showed intense green fluorescence, whereas no green fluorescence could be detected in neurons in which the
was driven by the 5′-flanking sequence of the human tRNA
Tyr.
Next we tested whether unnatural amino acids could be genetically encoded in neurons using the
and mutant synthetases that are specific for different unnatural amino acids. Synthetases that had been evolved in yeast and proven to be functional in HeLa cells were used. When the OmeTyrRS was coexpressed with the
, transfected neurons showed no green fluorescence in the absence of the corresponding unnatural amino acid OmeTyr (), indicating that the
is orthogonal to endogenous synthetases in neurons. Transfected neurons showed bright green fluorescence only when OmeTyr was fed to the growth medium. These results indicate that OmeTyr, but no common amino acid, is incorporated into GFP at the 182TAG position. The same results were obtained for the unnatural amino acid Bpa when the BpaRS was coexpressed with the
(). Using this approach, OmeTyr and Bpa were also genetically encoded in hippocampal and cortical neurons isolated from rats (data not shown).
Probing the inactivation mechanism of K+ channel Kv1.4
The fast inactivation of voltage-dependent K
+ (Kv) channels involves ~20 amino acids on the channel's intracellular N terminus, or on the N terminus of an associated β-unit. The first ~10 amino acids are predominantly hydrophobic, and the other ~10 amino acids are hydrophilic and positively charged
26. Recent crystal structure and mutation studies of Kv1.2 suggest that the inactivation peptide might snake through a side portal in the fully extended conformation, and allow the hydrophobic terminus to reach and plug the inner pore for inactivation
14-17 (). Through unnatural amino-acid mutagenesis of the inactivation peptide of Kv1.4 in live mammalian cells, we aimed to experimentally test this inactivation model by determining whether the bulkiness of the inactivation peptide would influence channel inactivation.
The N-terminal domain of Kv1.4 possesses two inactivation domains, consisting of amino acids Met1-Ala40 for the primary and Ala40-Gly60 for the secondary inactivation domains
27. The structure of the primary inactivation domain in aqueous solution has been analyzed using NMR spectroscopy
28,29. The N-terminal Met1-Met17 is disordered, followed by a β-turn (Pro18-Tyr21) and a well-defined α-helix (Tyr21-Ala36) (). In the Kv1.2 structure, a negatively charged patch of residues lies at the entrance to the side portal, and is proposed to interact with the positively charged amino acids on the inactivation peptide
14. According to this hypothesis, several arginines (Arg26, Arg28, Arg30 and Arg32) in the α-helix of Kv1.4 will interact with the negatively charged residues at the side portal entrance, which will place Tyr19 inside the side portal (). In addition, Tyr19 resides at the beginning of the α-helical portion of the inactivation domain (). We hypothesized that increasing the bulkiness of the amino-acid side chain at Tyr19 would alter the channel inactivation owing to the size restriction of the side portal.
We first investigated the inactivation properties of wild-type Kv1.4 channels expressed heterologously in human embryonic kidney 293 (HEK293) cells using whole-cell patch-clamp recordings (). A series of 1-s voltage steps from -100 mV to +40 mV elicited a rapidly inactivating macroscopic K
+ current. To avoid undesired transcriptional initiation at the downstream Met109 site and thus the generation of truncated Kv1.4, the methionine at 109 was mutated to leucine (M109L; see Methods). Kv1.4-M109L (Kv1.4
![[open star]](/corehtml/pmc/pmcents/star.gif)
) channels displayed fast inactivation similar to wild-type Kv1.4 channels (). To investigate the role of bulkiness in determining fast inactivation, we first mutated Tyr19 of Kv1.4
to Phe or Trp, using conventional site-directed mutagenesis. Compared to the native Tyr, Phe is smaller (lacking the hydroxyl) whereas Trp is considerably larger. The macroscopic K
+ currents recorded from HEK293 cells expressing Kv1.4
-Y19F or Kv1.4
-Y19W channels were similar to those of Kv1.4
channels (). Therefore, the Y19F or Y19W mutation did not greatly alter the inactivation properties.
Next, we tested whether a functional Kv1.4 channel could be expressed in HEK293 cells through amber suppression using the orthogonal
-TyrRs pair to incorporate the native Tyr. An amber stop codon (TAG) was introduced at Tyr19, and the mutant gene (
Kv1.4-
19TAG) was cotransfected into HEK293 cells with the
-TyrRs pair and the
GFP-
TAG reporter gene (). Transfected cells fluoresced green owing to the suppression of the amber codon in the
GFP-
TAG gene by the
-TyrRs pair. GFP-positive cells also showed large voltage-dependent K
+ currents that inactivated with large depolarizations (). To investigate possible differences in the rates of fast inactivation, we fit the decay of current elicited by the 1-s voltage-step to +20 mV with a sum of two exponentials relaxing to a small steady-state current (
C). Neither the τ
1 or τ
2 time constants nor the amplitudes of current were significantly different between Kv1.4
and Kv1.4
-19Tyr channels (; ), indicating that the incorporation of amino acids through amber suppression did not adversely affect the cell or the properties of the Kv1.4 channel. Importantly, no current could be detected in the absence of the
-TyrRs pair, confirming that functional Kv1.4 is produced through amber suppression and that the tRNA-synthetase pair is required for the incorporation of Tyr.
| Table 1Kinetics of current inactivation of Kv1.4 channels |
We then tested the effect of introducing a large bulky unnatural amino acid at position 19. OmeTyr was incorporated at the 19TAG position of Kv1.4
using the
-OmeTyrRS pair. When no OmeTyr was added to the growth medium, only small background levels of current could be detected in transfected cells (identified by cotransfected enhanced GFP in this case). By contrast, large voltage-gated K
+ currents were seen when OmeTyr was added to the culture medium (). Interestingly, the rate and extent of inactivation were markedly slower than with the wild-type channel — note the large amount of current at the end of the 1-s voltage step (). To quantify this change in inactivation more accurately, we measured the macroscopic current elicited by a 4-s voltage step to +20 mV and fit the decay of current with the sum of two exponentials, as described above (). The time constants of the fast (τ
1) and the slow inactivation component (τ
2) for the OmeTyr mutant increased 5- and 7-fold, respectively, compared to those of the wild-type Kv1.4
channel. In addition, the amplitude of the fast component (
A1) decreased to ~20%, whereas the amplitude of the slow component (
A2) increased by ~1.5-fold for the OmeTyr mutant (, ). We then tested the effect of incorporating an amino acid with an even larger side chain, DanAla, at Tyr19 (). As when OmeTyr was inserted, slowly inactivating, voltage-gated K
+ currents were seen in HEK293 cells transfected with Kv1.4
-19TAG, and the
-TyrRs pair, along with DanAla in the medium (). Quantification of the rate of inactivation showed that DanAla increased the fast and slow time constants for inactivation by 3.2-fold and 7-fold, respectively (, ). Similarly, the amplitude of the slow component was larger than that of the fast component of inactivation, in contrast to the wild-type channel. Thus, both OmeTyr and DanAla can be functionally incorporated into Kv1.4 channels, leading to markedly slower inactivation.
We also carried out several important controls. First, only small background currents were evident in HEK cells when the unnatural amino acid was omitted from the medium (). Thus, there was no inappropriate `read-through' of the amber mutation. To exclude the possibility that OmeTyr is toxic to cells or to the Kv channel, OmeTyr was also incorporated into Kv1.4
at 557TAG. Residue 557 is on the transmembrane helix S6 and lies at the inner pore, which is between the selectivity filter and intracellular solution. Like Kv1.4
, the inactivation rate of Kv1.4
-557OmeTyr was similar to that of wild-type Kv1.4 (; ). These experiments show that the abolition of fast inactivation of Kv1.4 by OmeTyr is site-dependent. To address whether the introduction of OmeTyr or DanAla rendered the N-terminal inactivation peptide subject to proteolytic cleavage, we added a FLAG tag to the C terminus of Kv1.4
-19TAG, and used western blotting to analyze channel proteins containing OmeTyr or DanAla at residue 19 expressed in HEK293 cells (). In both samples, one band corresponding to the full-length Kv1.4
channel was detected using FLAG-specific antibodies, indicating that there was no proteolytic cleavage accompanying the unnatural amino-acid incorporation.
The publisher's final edited version of this article is available at 
