dehydrogenase H carries a selenocysteine moiety. The codon (UGC) for this cysteine residue was changed into a UGA codon, and mutations were successively ...
Vol. 174, No. 3
JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 659-663 0021-9193/92/030659-05$02.00/0 Copyright X 1992, American Society for Microbiology
Targeted Insertion of Selenocysteine into the ca Subunit of Formate Dehydrogenase from Methanobacterium formicicum JOHANN HEIDER AND AUGUST BOCK* der Mikrobiologie Universitat Munchen, Maria-Ward-Strasse la, D-8000 Munich 19, Germany Lehrstuhlffur Received 27 September 1991/Accepted 5 November 1991
Selenocysteine incorporation into proteins is directed by an opal (UGA) codon and requires the existence of a stem-loop structure in the mRNA flanking the UGA at its 3' side. To analyze the sequence and secondary-structure requirements for UGA decoding, we have introduced mutations into the fdhA gene from Methanobacteriumformicicum, which codes for the a subunit of the F420-reducing formate dehydrogenase. The M. formicicum enzyme contains a cysteine residue at the position where the Escherichia coli formate dehydrogenase H carries a selenocysteine moiety. The codon (UGC) for this cysteine residue was changed into a UGA codon, and mutations were successively introduced at the 5' and 3' sides to generate a stable secondary structure of the mRNA and to approximate the sequence of the predicted E. colifdhF mRNA hairpin structure. It was found that introduction of the UGA and generation of a stable putative stem-loop structure were not sufficient for decoding with selenocysteine. Efficient selenocysteine incorporation, however, was obtained when the loop and the immediately adjacent portion of the putative stem had a sequence identical to that present in the E. coli fdhF mRNA structure. the fdhF gene from E. coli, especially in the neighborhood of the selenocysteine residue (25); it is important that the majority of the changes introduced should be silent. The fdhA gene product, which codes for the a subunit of the formate dehydrogenase of the methanogen, contains a cysteine (Cys-132) at this position, which is encoded by a UGC codon. We have converted this UGC into UGA, and we have consecutively introduced mutations into the upstream and downstream regions to achieve selenocysteine incorporation.
The incorporation of selenocysteine into selenoproteins is directed by an in-frame UGA codon in the mRNA (for reviews, see references 4 and 21). Analysis of the structural features of the mRNA of the fdhF gene from Escherichia coli, which codes for the 80-kDa selenopolypeptide of formate dehydrogenase H, has provided insight into the context requirements which differentiate this "sense" UGA codon (UGA-140) from a UGA termination codon (26). It was found that efficient readthrough required the presence of an mRNA segment of at least 40 bases downstream of the UGA; this sequence can be folded into a putative stem-loop structure. Deletions into this hairpin structure from the 3' side abolish the coding capacity of the UGA (26). A recent analysis of selenocysteine insertion into the 110-kDa selenopolypeptide of formate dehydrogenase N by Berg and coworkers has also shown that the process is dependent on the presence of a downstream sequence and secondary structure (2). Upstream sequences were shown not to be required for the specificity of incorporation, but they might be involved in the efficiency of the process, since deletions of upstream sequences close to the UGA reduce the rate of readthrough (26). One could envision a model for selenocysteine incorporation involving two cooperating mechanisms, namely (i) prevention of termination at the UGA and (ii) the decoding process itself. Distinct structural features of the selenoprotein mRNA may determine each of these functions. An experimental strategy which can be used to shed more light on this interesting and biologically basic question involves attempting to incorporate selenocysteine into a protein which, in the wild-type state, does not contain this amino acid. This "synthetic" approach will deliver information on the minimal number of sequence changes which must be made to convert a UGA codon into a code word for selenocysteine. We have chosen the fdhA gene from Methanobacterium formicicum (20) for this purpose, since its product has high amino acid sequence similarity with that of *
MATERIALS AND METHODS
Bacterial strains, phages, plasmids, media, and growth conditions. E. coli JM109 (24) was used throughout this study as the host for cloning and expression experiments. Strain CJ236 (dut ung) (11) was used in M13 mutagenesis work. WL308 is a Mudl deletion derivative of strain WL8 (12), which is MC4100 (5) carrying a Mudl insertion at the fdhD/E locus at min 88 of the E. coli chromosome (3; unpublished data). WL308 has lost the capacity to synthesize the 110-kDa selenoprotein subunit of formate dehydrogenase 0 (18). Phage M13mpl9 (17) was used as the cloning vector for mutagenesis experiments, which were performed as described by Kunkel et al. (11). Plasmid pUC19 (17) was used for expressing cloned genes in E. coli. Plasmid pUCFD18 (20), carrying the fdhAB genes from M. formicicum, was kindly provided by J. G. Ferry. E. coli cells were grown aerobically on LB medium (15) and anaerobically on TGYEP medium (pH 6.5) (1). For maximal expression of the cloned fdh genes from the lac promoter of plasmid pUC19, transformants were grown aerobically in medium containing 1% tryptone, 0.5% glycerol, 1 mM MgCl2, 0.1 mM CaCl2, 100 mM potassium phosphate (pH 7.0), 50 ,ug of ampicillin per ml, and trace elements as given by Neidhardt et al. (16). IPTG (isopropylthiogalactopyranoside, 1 mM) was present in the medium when strain JM109 was used and absent when strain WL308 was used in the experiments. Genetic procedures and recombinant DNA techniques. Standard recombinant DNA techniques were carried out as
Corresponding author. 659
660
HEIDER AND BOCK
described by Maniatis et al. (14). DNA fragments were recovered from agarose gels by the method of Vogelstein and Gillespie (23), and transformations were performed with the aid of the RbCl method (10). DNA nucleotide sequence determinations were carried out either by the chemical cleavage method (9) or by the chain termination procedure (6) with synthetic primers. Plasmid constructions. A 3.0-kb NcoI fragment from plasmid pUCFD18 (20), which carries the fdhA gene plus about one-third of the fdhB gene from M. formicicum, was cloned into the SmaI site of vector pUC19 so that the genes were under the control of the lac promoter. Cloning of the same restriction fragment into vector pT7-4 (22) and analysis of expression offdhAB' in E. coli by the method of Tabor and Richardson (22) revealed that only minute amounts of gene product were formed (data not shown). A construct was made to improve heterologous expression in E. coli. A 0.5-kb HindIII-SnaBI fragment containing the entire 5'flanking region of fdhA was replaced by a synthetic doublestranded oligonucleotide (JH7 annealed with JH8; for sequences, see below), resulting in a translational lacZ'-fdhA fusion. The resulting plasmid was then restricted with HindIII, and the recessive ends were filled in with Klenow enzyme; religation resulted in the generation of a UAG stop codon at the 3' end of the lacZ' portion of the fusion (see Fig. 3). The sequence of the oligonucleotide inserted was devised to provide an optimal ribosome-binding site. Furthermore, the second codon, GAT (Asp), of the fdhA reading frame was changed into a GCT (Ala) codon to conform with the situation found in highly expressed E. coli genes (8). The authenticity of the constructs was checked by DNA sequence analysis. The plasmid obtained in this way was designated pUGC. All mutated fragments were cloned into this plasmid. Introduction of mutations into fdhA. The TGC-132 codon of the M. formicicum fdhA gene was replaced by TGA by cloning a 1-kb NcoI-StuI fragment of plasmid pUCDF18 into EcoRV-HindII-cleaved vector pACYC184. Subsequently, a 30-bp ApaLI-BglI fragment carrying TGC-132 was replaced by a double-stranded synthetic oligonucleotide (JH1 annealed with JH2, see below) to generate a TGA-132 codon. The mutation was then transferred to the expression vector pUGC by exchanging SnaBI-BglII fragments. The plasmid generated was designated pUGA; the authenticity was checked by sequence analysis. All other mutations were introduced by the method of Kunkel et al. (11). To this end, a 1.4-kb HindIII-EcoRI fragment of pUGA was cloned into phage M13mpl9, and the recombinant phage was used as a source for single-stranded DNA. Degenerate mutagenic oligonucleotides (JH5, JH6, JH12, and JH13) were used to obtain phage lines with mutations in the regions flanking TGA-132 at the 5' (Fig. 1) or 3' (Fig. 2) side. Combinations of mutations were generated by using mutant phage clones obtained as templates for a second round of mutagenesis. The mutant DNA fragments were isolated from M13 replicative form DNA by restriction with endonucleases SnaBI and BglII and inserted into plasmid pUGC. Selenium incorporation experiments. Incorporation of radioactive selenium into macromolecules was performed by the method of Cox et al. (7). The medium used contained 0.5% (wt/vol) glycerol, 1% (wt/vol) tryptone, 100 mM potassium phosphate (pH 6.8), 5 ,M Na2MoO4, 50 pxg of ampicillin, and 0.5 ,uM [75Se]selenite (sodium salt) (specific activity, 400 mCi/mmol). Immunological procedures. Antibodies raised against puri-
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M. formbcum FIG. 1. Possible RNA secondary structures upstream of the UGC-132 codon in the M. formicicum fdhA gene and in those constructs mutated in the 5'-flanking region of UGA-132 of fdhA, compared with E. coli fdhF. Plasmids carrying the mutant variants were
designated appropriately (pUGC, pUGA, pUl, etc.).
fied M. formicicum formate dehydrogenase (20) were kindly provided by J. G. Ferry. They were used at a 1:2,000 dilution
for immunoblotting experiments, which were carried out as described before (19). Oligonucleotides. The sequences of the oligonucleotides used in this study are: JH1, CGGCTACAGTTGGGCCGT GTCAGAGTCG; JH2, TGCACGACTCTGACACGGCCCA
ACTGTAGCCGGAC; JH5, GTTGGGCCTGTCAGA(C/G) ACGAGCGCAGTGGTCAATGTTG; JH6, CCAACCAAA CGATGCGGCCAGTCCGGCCACAGTCGG(G/T)CCGTG TCAGA; JH7, AGCTTAGTTAACGGAGAAACAACTAT GGCTATTAAATAC; JH8, GTATTTAATAGCCATAGT TGTTTCTCCGTTAACTA; JH12, CGAACCGAACGACG GTGCAGACCTGCACCGTGGTCCGTGTCA; and JH13, CGAACCAAACGATCGGTGCAG(A/G)CCTGCAAC CGT CGGTCCGTGTCA.
RESULTS AND DISCUSSION In a previous communication, we have shown that insertion of selenocysteine into the formate dehydrogenase H selenopolypeptide requires the presence of a 40-base stretch at the 3' side of the UGA-140 codon in the fdhF mRNA (26). Deletions extending into this region abolished UGA-140 translation. A putative secondary structure (Fig. 2) can be formed in this mRNA segment; other alternative hairpin structures with the UGA-140 codon in the loop (Fig. 1) or at its 5' side (26) are also feasible. However, they were not essential for the specificity of selenocysteine insertion, since deletions approaching the UGA-140 codon from the 5' side affected UGA readthrough only quantitatively (26). Figures 1 and 2 show that the formation of similar structures is possible in the case of the fdhA mRNA sequence around UGC-132, but they are definitely less stable. In order to elucidate the structural features of the fdhA mRNA necessary for selenocysteine incorporation, we have followed the experimental approach depicted in Fig. 1 and 2: (i) the UGC-132 codon was altered into a UGA codon; (ii) base changes were introduced into the 5'-flanking sequence with the aim of generating a more stable form of the putative hairpin structure which carries the UGA in the loop (Fig. 1); (iii) mutations were introduced into the 3'-flanking region to stabilize the potential secondary structure downstream of the UGA; (iv) the sequence in the loop region was altered to
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VOL. 174, 1992
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generate a structure identical to that present in the E. coli fdhF mRNA (Fig. 2); and (v) the alterations introduced into the upstream region (Ul and U2 structures of Fig. 1) were combined with those introduced into the downstream region (D2, D21, and D22 of Fig. 2). For example, pU2D2 contains an fdhA gene with the sequence of the U2 variant upstream and that of the D2 variant downstream of UGA-132. The mutations generated were then transferred onto the expression plasmid pUGC (Fig. 3), the construction of which has been detailed under Materials and Methods. Transcription of the fdhA variants is under the control of the lac promoter indigenous to plasmid pUC19; an optimal ribosome-binding region at the correct distance precedes the different fdhA gene variants (Fig. 3). Figure 4 summarizes the sequence changes at the amino acid level created by the different mutations within the fdhA gene product. The plasmids carrying the mutant fdhA genes were transformed into E. coli WL308, the transformants were cultured in the presence of radioactive sodium selenite, and the radioactive macromolecules synthesized were analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography (Fig. 5). Lane 1 gives the position of the 80-kDa selenopolypeptide formed by wild-type cells under fermen-
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tative conditions, and lane 2 shows that under aerobiosis WL308, due to its mutation in the fdhDIE locus, does not synthesize any selenopolypeptide. A change of UGC-132 (lane 3) into UGA-132 (lane 4) is not sufficient to allow selenoprotein formation, even when "stabilizing" mutations are introduced into the region flanking the UGA codon at the 5' side (lanes 5 and 6). Mutations stabilizing the putative mRNA secondary structure at the downstream side of UGA132 also did not promote readthrough of the codon (lanes 7 through 10). An fdhA mutant (D21) was then constructed which is characterized by having a somewhat chimeric stem-loop structure 3' to UGA-132, the lower part of the stem being identical to that of construct D2 and the upper part (boxed in Fig. 2) resembling the E. coli structure with the exception of a C (circled) in the loop region instead of a U. WL308 carrying this fdhA gene incorporated a small amount of labeled selenium into a macromolecule migrating at the position expected for the full-length fdhA gene product (lanes 12 and 13). This C was then subsequently altered into a U residue (construct D22), and it was found that the amount of selenylated putative a subunit was drastically increased (lanes 14 and 15).
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HEIDER AND BOCK
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FIG. 5. [75Se]selenium incorporation by E. coli WL308 harboring plasmids carrying the M. formicium fdhA gene and mutated derivatives thereof (see Fig. 2 and 3). Lanes: 1, MC4100 grown anaerobically as a control for the 80-kDa selenopolypeptide of formate dehydrogenase H; 2 through 15, WL308 grown aerobically and containing plasmids pUC19, pUGC, pUGA, pUl, pU2, pDl, pD2, pUlD2, pU2D2, pD20, pD21, pU2D21, pD22, and pU2D22, respectively. The structure of Dl is not shown in Fig. 2. It differs from D2 in that the A residue six nucleotides downstream of the UGA-132 is a C.
In construct D20, the looped-out U residue was removed and the reading frame was adjusted by deletion of two bases from the lower part of the stem. As a consequence, selenium incorporation was abolished (lane 11). By immunoblotting experiments with antibodies directed against purified formate dehydrogenase from M. formicicum (20), it was analyzed whether the selenium incorporation pattern observed correlated with the formation of immunoreactive material. Figure 6 demonstrates that this is indeed the case; a faint signal was seen when extracts were separated from a strain carrying the fdhA gene variant D21 (lanes 12 and 13), and massive amounts were detected with construct D22. In none of the variants tested did a change in the "upstream" region (Ul or U2) alter the effects caused by "downstream" changes. The immunoreactive material of a size lower than the mature 93-kDa gene product was present in all of the extracts carryingfdhA genes on the plasmid and is presumably due to translational reinitiation downstream of UGA-132. To analyze whether a functional M. formicicum formate dehydrogenase can be formed and assembled in the heterologous host E. coli, we have completed the fdhAB operon in pUGC and the variant D22 by adding the fdhB gene (data not
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FIG. 6. Immunoblotting analysis of expression of fdhA and its mutated derivatives in E. coli JM109 after aerobic growth in the presence of 1 mM IPTG. Lanes 1 through 15, JM109 containing
pUC19, pUGC, pUGC, pUGA, pUl, pU2, pDl, pD2, pUlD2, pU2D2, pD20, pD21, pU2D21, pD22, and pU2D22, respectively. Lane 2 contains half the amount of protein present in the other lanes.
shown). The plasmids were transferred into E. coli FM911 (AfdhF), and the transformants were analyzed for formatecoupled benzyl viologen reduction upon growth under anaerobic conditions (13). The transformants were unable to reduce the dye. We have not analyzed whether this inability is due to their inability to incorporate any of the cofactors or coenzymes (Zn, Fe/S, molybdopterin, flavin adenine dinucleotide) (20), to the amino acid sequence changes present, or to some other feature of the constructs. In conclusion, we have provided the first example of a targeted insertion of selenocysteine instead of a cysteine residue into a protein. As expected from previous results (26), substitution of the cysteine codon by a UGA codon was not sufficient to direct incorporation. A further important result was that creation of a stable secondary structure downstream of the UGA also did not meet the requirements for decoding. Rather, the data indicate that specific sequences in the loop region and in the adjacent part of the putative hairpin structure are necessary. Intensive localized mutagenesis experiments support this conclusion (unpublished results) and suggest that this part of the mRNA interacts with some other component of the translational system. ACKNOWLEDGMENTS We are greatly indebted to J. G. Ferry for providing plasmid pUCDF18 and antiserum directed against M. formicicum formate dehydrogenase. We thank G. Arnold for the synthesis of oligonucleotides. This work was supported by the Bundesministerium fur Forschung und Technologie via Genzentrum Munchen and the Fonds der Chemischen Industrie. REFERENCES
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5:1-10. 20. Shuber, A. P., E. C. Orr, M. A. Recny, P. F. Schendel, H. D. May, N. L. Schauer, and J. G. Ferry. 1986. Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacteriumformicicum. J. Biol. Chem. 261:1294212947. 21. Stadtman, T. C. 1990. Selenium biochemistry. Annu. Rev. Biochem. 59:111-127. 22. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074-1078. 23. Vogelstein, B., and D. Gillespie. 1979. Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76:615-619. 24. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-109. 25. Zinoni, F., A. Birkmann, W. Leinfelder, and A. Bock. 1987. Cotranslational insertion of selenocysteine into formate dehydrogenase from Escherichia coli directed by a UGA codon. Proc. Natl. Acad. Sci. USA 84:3156-3160. 26. Zinoni, F., J. Heider, and A. Bock. 1990. Features of the formate dehydrogenase mRNA necessary for decoding of the UGA codon as selenocysteine. Proc. Natl. Acad. Sci. USA 87:46604664.
