Journal of Bacteriology, June 2003, p . 3524-3526, Vol . 185, No . 12

Protein Synthesis in Escherichia coli with Mischarged tRNA

Bokkee Min,¹ Makoto Kitabatake,¹ Carla Polycarpo,¹ Joanne Pelaschier,¹ Gregory Raczniak,¹ Benfang Ruan,¹ Hiroyuki Kobayashi,¹ Suk Namgoong,¹ and Dieter Söll^1,2*

Departments of Molecular Biophysics and Biochemistry,¹ Chemistry, Yale University, New Haven, Connecticut 06520-8114²

Received 17 December 2002/ Accepted 2 April 2003

 
Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA^Asp, while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA^Asn, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli) . On the basis of the E . coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA^Asn and its acceptance by elongation factor EF-Tu . While large amounts of Asp-tRNA^Asn are detrimental to E . coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase .

 
Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition (7) . The discriminating enzyme (D-AspRS) recognizes only tRNA^Asp, while the nondiscriminating one (ND-AspRS) also recognizes tRNA^Asn and therefore forms both Asp-tRNA^Asn and Asp-tRNA^Asp . Most bacteria and archaea lack asparaginyl-tRNA synthetase and are unable to synthesize Asn-tRNA^Asn by direct acylation of tRNA . These organisms rely on the ND-AspRS to produce the misacylated Asp-tRNA^Asn, which is then converted by a tRNA-dependent amidotransferase to the correctly acylated Asn-tRNA^Asn (1, 4, 5, 19) . Thus, the ND-AspRS is essential in organisms that form Asn-tRNA by transamidation .

The primary sequence distinguishes two general groups of AspRS . There is a predominantly bacterial type of AspRS that is about 580 amino acids, in addition to a shorter archaeal-eukaryotic type of about 430 amino acids . In vitro data have made clear that discriminating and nondiscriminating enzymes exist in both groups (16, 20) . The determinants in the protein sequence responsible for tRNA discrimination are not known .

The two AspRS types are usually separated in nature . Genome analyses of bacteria and archaea have revealed that the presence of the ND-AspRS is always accompanied by the occurrence of the heterotrimeric GatCAB amidotransferase, an enzyme capable of converting the misacylated Asp-tRNA^Asn to Asn-tRNA^Asn (2, 5, 19) . Presumably, this is to avoid introducing the misacylated Asp-tRNA^Asn into an organism's translational apparatus and potentially endangering protein synthesis . This reasoning is supported by the fact that the heterologous expression of ND-AspRS or ND-GluRS in Escherichia coli, which lacks GatCAB, is highly toxic to the cell, especially when the synthetase genes are overexpressed (15) . However, some organisms (e.g., Deinococcus radiodurans and Thermus thermophilus) contain a D-AspRS in addition to an ND-AspRS and a GatCAB amidotransferase (1, 3, 5, 9) .

We wanted to observe how E . coli copes with in vivo mischarging effected by the ND-AspRS, as this organism is unable to eliminate the toxic Asp-tRNA^Asn . Therefore, we developed an approach that would, in fact, require E . coli to be dependent on the presence of mischarged Asp-tRNA^Asn for growth . To this aim, we used missense suppression of a specific mutation in the trpA gene brought about by transformation of E . coli with the genes of several different ND-AspRS enzymes .

 
Plasmids and strains. AspRS genes were cloned into pCR2.1-TOPO (Invitrogen), while aspS complementation studies were carried out with pCBS1 (6) and pBAD-TOPO (Invitrogen) . Expression of the desired gene in the latter vector is induced by arabinose . E . coli DH5 was used for most of the cloning experiments . E . coli trpA34 strains (17) carrying a D60N mutation in trpA were used in missense suppression tests . E . coli strain A2/A2 (10) was used for synthesis of indole-3-glycerol phosphate (IGP), the substrate for the tryptophan synthetase assay .

AspRS enzymes used. The standard bacterial-type D-AspRS in our experiments was the E . coli enzyme (11). D . radiodurans provided both a larger D-AspRS1 and a small ND-AspRS2 (9) . The Chlamydia trachomatis ND-AspRS resembling the standard bacterial enzyme (16) was used, as well as the Halobacterium salinarum archaeal-type ND-AspRS (accession no. BAA20527) .

Plasmids carrying AspRS and tRNA^Asn genes. With genomic DNA, the aspS genes (from the start codon to the stop codon) were PCR amplified and cloned into the pCR2.1-TOPO or pBAD-TOPO vector . After sequence confirmation, they were recloned into the pCBS1 vector behind the trpS promoter for low-level constitutive expression . The H . salinarum tRNA^Asn gene was constructed from two oligonucleotides (91 and 94 nt) inserted between the lpp promoter and the rrn terminator of the chloramphenicol resistance-encoding pTECH vector, derived from pGFIB (13) and pACYC184 by Tong Li and Makoto Kitabatake (Yale University) .

Suppression of the E . coli trpA34 strain. The trpA34 strain was transformed with each of the pCBS1 (for low-level expression) and pBAD-TOPO (for high-level expression) plasmids containing aspS genes from the sources mentioned previously . Ampicillin-resistant colonies were streaked onto M9 minimal agar plates supplemented with 19 amino acids (20 µg/ml) in the presence or absence of tryptophan (20 µg/ml), incubated at 37°C for 5 days, and scored daily .

Tryptophan synthetase assay. Freshly grown seed cultures in Vogel-Bonner minimal medium with or without Trp (20 µg/ml) were inoculated into 500 ml of the same medium . The cultures were grown at 37°C to late log phase, harvested by centrifugation, washed twice with ice-cold NaCl (0.9%) solution, and resuspended in buffer A (0.05 M KPO₄ [pH 7.0], 0.1 mg of pyridoxal-5-phosphate per ml, 10 mM 2-mercaptoethanol) . Cell extracts were prepared (18), dialyzed against buffer A containing 50% glycerol, and stored at -20°C . IGP was freshly prepared as described by Mosteller (10) . Tryptophan synthetase was assayed in the IGPTrp conversion with [³H]Ser (28.0 Ci/mmol) and [¹⁴C]Trp (58.1 mCi/mmol) (18) .

 
Missense suppression of trpA34. The E . coli trpA34 mutation is a GATAAT change in codon 60 of the trpA gene (17); the resulting DN alteration causes loss of the catalytically essential D60 residue in the subunit of tryptophan synthetase and leads to enzyme inactivation . As a consequence, the E . coli trpA34 mutant strain is a Trp auxotroph (17) . However, the presence in E . coli of mischarged Asp-tRNA^Asn should lead to reinsertion of D at the AAU codon (specifying N) and enable synthesis of wild-type tryptophan synthetase and restoration of prototrophic growth . This should provide a sensitive test for the presence of Asp-tRNA^Asn and allow in vivo examination of the tRNA recognition properties of AspRS enzymes .

Asp-tRNA^Asn formation in vivo. The ability of the ND-AspRS enzymes from C . trachomatis, H . salinarum, and D . radiodurans to form the missense suppressor Asp-tRNA^Asn in vivo in E . coli was tested by transforming the trpA34 mutant strain with the relevant cloned aspS genes . The results summarized in Table 1 show that the E . coli trpA34 mutant strain transformed with the empty vector or with D . radiodurans aspS1 did not grow on minimal medium lacking Trp . However, the D . radiodurans aspS2 gene (cloned in pCBS1) supported growth in minimal medium (Table 1) but C . trachomatis and H . salinarum aspS did not . While increased expression of C . trachomatis aspS (the pBAD-TOPO transformant in the absence of arabinose) allowed growth on minimal medium, H . salinarum aspS suppressed trpA34 only when the H . salinarum tRNA^Asn gene was also expressed in the E . coli strain (Fig . 1 and Table 1) . This indicates that the H . salinarum AspRS does not recognize E . coli tRNA^Asn but charges the RNA product of the homologous H . salinarum tRNA^Asn gene expressed in E . coli . Under the conditions described above, the C . trachomatis aspS transformant grew best on minimal medium while the strains transformed with D . radiodurans aspS2 and H . salinarum aspS grew two and three times slower, respectively .

 
We then proceeded to measure tryptophan synthetase activity in the cell extracts of the transformed strains (Table 2) . As expected, E . coli aspS (the empty-vector control) and D . radiodurans aspS1 did not confer any tryptophan synthetase activity . However, the three ND-aspS genes all gave rise to sizable tryptophan synthetase activities, i.e., up to 38% of the amount measured in the wild-type E . coli W3110 strain . This suggests that if the observed levels of tryptophan synthetase (Table 2) are a consequence of the amount of Asp-tRNA^Asn formed in E . coli by the heterologous ND-AspRS enzymes, then the higher levels of the mischarged tRNA may be correspondingly more toxic to the cell because of a certain level of general misincorporation of aspartate specified by asparagine codons . Therefore, it is reasonable that the trpA34 mutant strain transformed with the H . salinarum AspRS and tRNA^Asn displayed the slowest growth .

 
These results raise a number of questions . What levels of mischarged tRNA can a cell tolerate? The phenomenon of missense suppression (12, 14) mandates that a cell can cope with a small level of mischarging . However, this has never been investigated in detail . Furthermore, it is assumed that misacylated tRNA is discriminated against by elongation factor EF-Tu (1) . While this is supported by elegant biochemical studies (8), the levels of discrimination in vivo have not been established . It may also be possible that the properties with EF-Tu in this regard may vary depending on the whether or not the organism synthesizes amide aminoacyl-tRNAs by the transamidation route . Additionally, the concentrations of correctly acylated versus misacylated tRNA may affect the discrimination process . Future genetic experiments based on the trpA34 system should further our knowledge of specificity in the process of protein biosynthesis .

 
We are indebted to Fran Pagel and Emmanuel Murgola for strains and advice .

This work was supported by grants from the Department of Energy, the National Aeronautics and Space Administration, and the National Institute of General Medical Sciences .

 
* Corresponding author . Mailing address: Department of Molecular Biophysics and Biochemistry, Yale University, P.O . Box 208114, 266 Whitney Ave., New Haven, CT 06520-8114 . Phone: (203) 432-6200 . Fax: (203) 432-6202 . E-mail: soll@trna.chem.yale.edu .

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