Protein Synthesis in Escherichia coli with Mischarged tRNA

Two types of aspartyl-tRNA synthetase exist: the discriminatingenzyme (D-AspRS) forms only Asp-tRNA^Asp, while the nondiscriminatingone (ND-AspRS) also synthesizes Asp-tRNA^Asn, a required intermediatein protein synthesis in many organisms (but not in Escherichiacoli). On the basis of the E. coli trpA34 missense mutant transformedwith heterologous ND-aspS genes, we developed a system withwhich to measure the in vivo formation of Asp-tRNA^Asn and itsacceptance by elongation factor EF-Tu. While large amounts ofAsp-tRNA^Asn are detrimental to E. coli, smaller amounts supportprotein synthesis and allow the formation of up to 38% of thewild-type level of missense-suppressed tryptophan synthetase.

INTRODUCTION

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Introduction

Aspartyl-tRNA synthetase (AspRS) exists in two different formswith respect to tRNA recognition (7). The discriminating enzyme(D-AspRS) recognizes only tRNA^Asp, while the nondiscriminatingone (ND-AspRS) also recognizes tRNA^Asn and therefore forms bothAsp-tRNA^Asn and Asp-tRNA^Asp. Most bacteria and archaea lackasparaginyl-tRNA synthetase and are unable to synthesize Asn-tRNA^Asnby direct acylation of tRNA. These organisms rely on the ND-AspRSto produce the misacylated Asp-tRNA^Asn, which is then convertedby a tRNA-dependent amidotransferase to the correctly acylatedAsn-tRNA^Asn (1, 4, 5, 19). Thus, the ND-AspRS is essential inorganisms 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 about580 amino acids, in addition to a shorter archaeal-eukaryotictype of about 430 amino acids. In vitro data have made clearthat discriminating and nondiscriminating enzymes exist in bothgroups (16, 20). The determinants in the protein sequence responsiblefor tRNA discrimination are not known.

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

We wanted to observe how E. coli copes with in vivo mischargingeffected by the ND-AspRS, as this organism is unable to eliminatethe toxic Asp-tRNA^Asn. Therefore, we developed an approach thatwould, in fact, require E. coli to be dependent on the presenceof mischarged Asp-tRNA^Asn for growth. To this aim, we used missensesuppression of a specific mutation in the trpA gene broughtabout by transformation of E. coli with the genes of severaldifferent ND-AspRS enzymes.

MATERIALS AND METHODS

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Materials and Methods

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

AspRS enzymes used. The standard bacterial-type D-AspRS in our experiments was theE. coli enzyme (11). D. radiodurans provided both a larger D-AspRS1and a small ND-AspRS2 (9). The Chlamydia trachomatis ND-AspRSresembling the standard bacterial enzyme (16) was used, as wellas the Halobacterium salinarum archaeal-type ND-AspRS (accessionno. BAA20527).

Plasmids carrying AspRS and tRNA^Asn genes. With genomic DNA, the aspS genes (from the start codon to thestop codon) were PCR amplified and cloned into the pCR2.1-TOPOor pBAD-TOPO vector. After sequence confirmation, they wererecloned into the pCBS1 vector behind the trpS promoter forlow-level constitutive expression. The H. salinarum tRNA^Asngene was constructed from two oligonucleotides (91 and 94 nt)inserted between the lpp promoter and the rrn terminator ofthe chloramphenicol resistance-encoding pTECH vector, derivedfrom 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 (forlow-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 minimalagar plates supplemented with 19 amino acids (20 µg/ml)in the presence or absence of tryptophan (20 µg/ml), incubatedat 37°C for 5 days, and scored daily.

Tryptophan synthetase assay. Freshly grown seed cultures in Vogel-Bonner minimal medium withor without Trp (20 µg/ml) were inoculated into 500 mlof the same medium. The cultures were grown at 37°C to latelog phase, harvested by centrifugation, washed twice with ice-coldNaCl (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 Acontaining 50% glycerol, and stored at -20°C. IGP was freshlyprepared as described by Mosteller (10). Tryptophan synthetasewas assayed in the IGP $->$ Trp conversion with [³H]Ser (28.0 Ci/mmol)and [¹⁴C]Trp (58.1 mCi/mmol) (18).

RESULTS AND DISCUSSION

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Results and Discussion

Missense suppression of trpA34. The E. coli trpA34 mutation is a GAT $->$ AAT change in codon 60 ofthe trpA gene (17); the resulting D $->$ N alteration causes lossof the catalytically essential D60 residue in the ${alpha}$ subunit oftryptophan synthetase and leads to enzyme inactivation. As aconsequence, the E. coli trpA34 mutant strain is a Trp auxotroph(17). However, the presence in E. coli of mischarged Asp-tRNA^Asnshould lead to reinsertion of D at the AAU codon (specifyingN) and enable synthesis of wild-type tryptophan synthetase andrestoration of prototrophic growth. This should provide a sensitivetest for the presence of Asp-tRNA^Asn and allow in vivo examinationof 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 suppressorAsp-tRNA^Asn in vivo in E. coli was tested by transforming thetrpA34 mutant strain with the relevant cloned aspS genes. Theresults summarized in Table 1 show that the E. coli trpA34 mutantstrain transformed with the empty vector or with D. radioduransaspS1 did not grow on minimal medium lacking Trp. However, theD. radiodurans aspS2 gene (cloned in pCBS1) supported growthin minimal medium (Table 1) but C. trachomatis and H. salinarumaspS did not. While increased expression of C. trachomatis aspS(the pBAD-TOPO transformant in the absence of arabinose) allowedgrowth on minimal medium, H. salinarum aspS suppressed trpA34only when the H. salinarum tRNA^Asn gene was also expressed inthe E. coli strain (Fig. 1 and Table 1). This indicates thatthe H. salinarum AspRS does not recognize E. coli tRNA^Asn butcharges the RNA product of the homologous H. salinarum tRNA^Asngene expressed in E. coli. Under the conditions described above,the C. trachomatis aspS transformant grew best on minimal mediumwhile the strains transformed with D. radiodurans aspS2 andH. salinarum aspS grew two and three times slower, respectively.

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TABLE 1. Growth of trpA34 strains containing aspS genes from D. radiodurans, C. trachomatis, or H. salinarumin the absence of tryptophan^a

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FIG. 1. Growth of E. coli trpA34 mutant strains transformed with aspS genes from D. radiodurans, C. trachomatis, and H. salinarum on minimal agar plates in the absence (-Trp) or presence (+Trp) of tryptophan. —, empty pCBS1 vector; DR1, D. radiodurans aspS1 in pCBS1; DR2, D. radiodurans aspS2 in pCBS1; CT, C. trachomatis aspS in pBAD-TOPO; HS, H. salinarum aspS in pBAD-TOPO plus tRNA^Asn in pTECH. The picture was taken after 5 days of incubation at 37°C.

We then proceeded to measure tryptophan synthetase activityin the cell extracts of the transformed strains (Table 2). Asexpected, E. coli aspS (the empty-vector control) and D. radioduransaspS1 did not confer any tryptophan synthetase activity. However,the three ND-aspS genes all gave rise to sizable tryptophansynthetase activities, i.e., up to 38% of the amount measuredin the wild-type E. coli W3110 strain. This suggests that ifthe observed levels of tryptophan synthetase (Table 2) are aconsequence of the amount of Asp-tRNA^Asn formed in E. coli bythe heterologous ND-AspRS enzymes, then the higher levels ofthe mischarged tRNA may be correspondingly more toxic to thecell because of a certain level of general misincorporationof aspartate specified by asparagine codons. Therefore, it isreasonable that the trpA34 mutant strain transformed with theH. salinarum AspRS and tRNA^Asn displayed the slowest growth.

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TABLE 2. Tryptophan synthetase activities in W3110 and trpA34 strains containing AspRSs from D. radiodurans, C. trachomatis, and H. salinarum grown in the absence of tryptophan

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

ACKNOWLEDGMENTS

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

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

FOOTNOTES

* 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{at}trna.chem.yale.edu.

REFERENCES

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