Molec. gen. Genet. 170, 231-234 (1979) © by Springer-Verlag 1979
Short Communication
Cloning of an EcoRI Fragment Carrying E. coil tufA Gene Masabumi Shibuya, Hiroko Nashimoto, and Yoshito Kaziro Institute of Medical Science, University of Tokyo, Takanawa, Minatoku, Tokyo 108, Japan
Summary. EcoRI fragments of the transducing phage 2fus3 DNA have been linked to the ColE1 derivative plasmid RSF2124 (ColE1-Ap r) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUA1 was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUA1 is based on the following observations: (1) ability of pTUA1 DNA and its EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) R N A . D N A hybridization of RNA transcribed from phage 2rlfdl 8 carrying tufB with DNA from pTUA1.
Polypeptide chain elongation factor Tu promotes a GTP-dependent binding of aminoacyl-tRNA to ribosomes. The protein has a molecular weight of 47,000, and its properties, structure and function have been extensive!y studied (for recent reviews, see Miller and Weissbach, 1977; and Kaziro, 1978). It is present in E. coli cells relatively in a large quantity (Gordon, 1970) and its level under balanced growth increases as cells grow more rapidly (Furano and Wittel, 1976; Reeh et al., 1976; Blumenthal et al., 1976; Miyajima and Kaziro, 1978), The genetic element for EF-Tu is located in two distinct loci on the E. coli chromosome (Jaskunas et al., 1975); one at 72min (tufA) and the other at 88 rain (tufB) on a recalibrated map of E. coli (Bachmann et al., 1976). The transducing phages containing tufA and tufB have been isolated and their fine genetic organizations have been established (Nomura, Morgan and Jaskunas, 1977). In this paper, we report cloning of the tufA gene from 2fus3 DNA into ColE1 derivative plasmid RSF2124 (ColE1Ap r) creating a new hybrid plasmid designated pTUA1. Some properties of the cloned DNA are also described. For offprints contact: M. Shibuya
2fus3 DNA was prepared according to Miller (1972) from phage 2cI857S7fus3 produced by the heat-induced culture of NO1380 (2K401-3RrecA (2cI857S7xis665156519) (2fus3)). The DNA was dialyzed against, and stored in, 10 mM Tris-HCl (pH 7.5) containing 10 mM MgC12, 100 mM NaC1 and 10 mM dithiothreitol (EcoRI buffer). RSF2124 DNA was isolated from the cleared lysate of TM201 (E. coli K12, thy, trp, str~(RSF2124) (So, Gill, and Falkow, 1975)) by phenol extraction, Bio-Rad A5m gel filtration, and further purified by two cycles of ethidium bromide-CsC1 density equilibrium centrifugation, followed by dialysis against EcoRI buffer. For construction of the recombinant DNA molecule of RSF2124 containing an EcoRI fragment carrying tufA, a mixture of 4 ~g of RSF2124 DNA and 8 gg of 2fus3 DNA was digested by EcoRI endonuclease at 37° C. After heating for 10rain at 70° C, 0.1 mM ATP (final concentration), and an excess of T4 ligase were added to the mixture, and the incubation was further continued for 4 h at 10° C. After heating again for 10 rain at 70° C, the reaction mixture was directly used to transform cells of E. coli C600 (thr, leu, thi, lac) grown in PC medium (Cameron et al., 1975). Ampicilin-resistant transformants which did not produce colicin were selected. Since tufA is located on the 8.5% EcoRI fragment of transducing phage )fus3 DNA (Jaskunas et al., 1977; Lindahl et al., 1977), we surveyed the plasmid DNA of about 50 transformants obtained as above, and found that 5 transformants contained about 8.5 to 9% EcoRI fragments cloned in RSF2124. Among 5 such clones, one designated pTUAI was found to contain the 8.5% EcoRI fragment (Fig. 1A slot 1). The presence of the 8.5% EcoRI fragment from )fus3 DNA in pTUA1 DNA was further confirmed by its cleavage pattern with Sma. As shown in Fig. 1 C, the fragment possesses 4 Sma sites and the digestion of the fragment with Sma would yield 5 fragments, i.e. 0.9, 0.6, 2.1 a, 2.1 b, and 2.3%-2units. 0026-8925/79/0170/0231/$01.00
232
M. Shibuya et al. : Cloning of an EcoRI Fragment Carrying E. coli tufa Gene
A
B
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C 0.9% 0.6 Eco R 1 Sm(3 /
sm\2 .oz/ ......
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Fig. 1A-C. Agarose gel electrophoresis of endonuclease-digested pTUA1 and 2fus3 DNA and the orientation of the tufA-containing EcoRI fragment in pTUAI DNA. About 5-10 ~tg of DNA was digested with EcoRI,or Srna, and at the end of digestion, the reaction mixture was supplemented with one-sixth volume of the solution containing 60 mM Na2EDTA, 0.6% sodium dodecylsulfate (SDS), 0.6% bromphenol blue and 60% glycerol, and was applied onto a 4 ram-thick 0.8% agarose slab gel. Electrophoreses were performed in a buffer containing 40 mM Tris-acetate (pH 7.4), 20 mM sodium acetate, 2 mM NazEDTA and 0.5 ~tg/ml ethidium bromide for 5 to 6 hrs at 3 V/cm. (A) slot 1, pTUA1 DNA digested with EcoRI; and slot 2, )~fus3 DNA digested with EcoRI. The arrow indicates the position of EeoRI-digested RSF2124 DNA. (B) slot 1, pTUA1 DNA digested with Sma; and slot 2, 2fus3 DNA digested with Sma. The numbers to the right of (A) and (B) indicate the size of 2fus3 fragments in %-2units. The numbers in parentheses are fragments derived from the cohesive end of 2DNA. (C) Orientation of the 8.5% EcoRI fragment within pTUAI DNA showing the cleavage sites of endonucleases EcoRI and Sma. The alignment of Sma fragments in the 8.5 % EcoRI fragment is based on the observations of Jaskunas et al. (1977), Lindahl et al. (1977), and Furano (1978). More recently, we have proved this unequivocally by comparing the sequence of tufa DNA and that of EF-Tu protein (Yokota et al., unpublished)
Now, the analysis of Sma fragments of pTUA1 DNA revealed the presence of three distinct bands, 21.5, 4.7, and 2.1% fragments, on agarose gel electrophoresis (Fig. 1B slot 1). The 2.1% Sma fragment is characteristic to the 8.5% EcoRI fragment of 2fus2(3) (Nomura, 1977). The 0.6% Srna fragment was not detected in Fig. 1 B, but was clearly observed by 3.5% acrylamide-0.1% bisacrylamide gel electrophoresis (data not shown). Plasmid RSF2124 DNA (23%-2unit) has one Sma site in its colicin E1 gene, at about 2.4%-)mnit from the EcoRI site (Dougan et al., 1978). The appearance of 4.7 (2.3+2.4) and 21.5 (0.9+20.6)% Srna fragments in the Sma digestion pattern of pTUA1 DNA indicates that the 8.5% EcoRI fragment is oriented in pTUA1 DNA as illustrated in Fig. 1 C. To confirm that the entire structural gene for EFTu is cloned in the plasmid pTUA1, we analyzed proteins synthesized in the cell-free system under the direction of pTUA1 DNA or its fragments produced by cleavage with EcoRI or Sma restriction endonuclease. The DNA-dependent protein-synthesizing system used in the present study was similar to Zubay
et al. (1970) but was modified as follows. The $30 fraction derived from E. coli PR13 was treated with polyethylene glycol-6000 to remove endogenous DNA. The DNA-free polyethylene glycol-treated extracts were then supplemented with unwashed ribosomes. The incorporation of labeled amino acids in this system was completely dependent on the presence of added DNA. The details of the procedure will be published elsewhere. As shown in Fig. 2, when intact pTUA1 DNA was utilized as a template, the proteins formed contained several bands migrating close to authentic EFTu on SDS-polyacrylamide gel electrophoresis (slot 1). On digestion of the template with EcoRI, the radioactivity in the EF-Tu band was much increased to become a predominant band (slot 2). Since the natural promoter for tufA transcription is not located on the 8.5% EcoRI fragment (Jaskunas et al., 1977), the creation of some artificial initiation site by EcoRI digestion might be responsible for the increased synthesis of EF-Tu. As expected, no band corresponding to EF-Tu was seen after digestion of pTUA1 DNA with Sma (slot 3) and also in the control
M. Shibuya et al. : Cloning of an EcoRI Fragment Carrying E. coli tufA Gene
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Fig. 2. SDS-polyacrylamide gel electrophoresis of proteins synthesized in the cell-free system using intact or restriction endonuclease digested pTUA1 DNA as a template. Proteins were synthesized in a cell-free system prepared as described in the text. The reaction mixture contained in a final volume of 0.1 ml; 40 mM Tris-acetate (pH 8.2), 60 mM potassium acetate, 30 mM NHgC1, 15 mM magnesium acetate, 1.5 mM dithiothreitol, 3% (w/v) polyethylene glycol-6000, 0.5 mM each CTP, UTP, and GTP, 2 mM ATP; 0.7 gg ofpyruvate kinase, 20 mM phosphoenolpyruvate, 6 gg of folinic acid; 40 gM of [14C]leucine (340 I,tCi/~tmol), 15 gM of [14C]phenylalanine (420 gCi/gmol), and 0.2 mM each of other 18 amino acids; 120 gg of polyethylene glycol-treated $30 proteins, 2 Az60 units of unwashed ribosomes; 20 gg of uncharged tRNA, and 4 gg of DNA used as a template. The components were mixed at 0°C and incubated for 60min at 37* C. An aliquot (15 gl) was taken from each reaction mixture, and the radioactive proteins formed was electrophoresed on the slab gels containing 7.5% acrylamide - 0.2% bisacrylamide and 0.1% SDS (Weber and Osborn, 1969). The templates used are: (1) intact pTUA1 DNA, (2) EcoRIdigested pTUA1 DNA, (3) Sma-digested pTUA1 DNA, (4) intact RSF2124 DNA, (5) EcoRI-digested RSF2124 DNA, and (6) Smadigested RSF2124 DNA. Position of EF-Tu was indicated by an arrow. The amount of EF-Tu synthesized in each sample was roughly estimated using anti-EF-Tu antibody and was shown in the bottom of each slot using a symbol of ( - ) , nondetectable; (+), detectable; and (+ + +), abundant, Anti-EF-Tu immunoglobulin was a gift from Mr. A. Miyajima of this laboratory
system directed by either an intact (slot 4) or EcoRItreated (slot 5) vehicle RSF2124 DNA. For further confirmation of pTUA1 DNA-directed synthesis of EF-Tu, specific immunoprecipitation with anti-EF-Tu antibody and gel electrophoresis of the immune-precipitates were carried out according to Miyajima and Kaziro (1978). The results clearly indicated that EF-Tu was synthesized in the presence of pTUA1 DNA or its EcoRI digested fragments, and the amount of EF-Tu synthesized was severalfold higher in the latter as compared to the former (data not shown).
233
It has been shown by Jaskunas et al. (1975) that another gene for EF-Tu, i.e. tufB, is encoded in transducing phage 2r/fal8 (Kirschbaum and Konrad, 1973). Recent evidence indicates that the proteins produced by tufa and tufB are very similar, if not identical, in terms of their molecular weight, isoelectric point, pattern of tryptic digestion, and catalytic properties (Jaskunas et al., 1975; Furano, 1977; Miller et al., 1978). Therefore, it is expected that tufB DNA as well as its in vitro RNA transcripts would form a hybrid with pTUA1 DNA. To test this possibility, we have synthesized 3H-labeled RNA using E. coli RNA polymerase holoenzyme under the direction of 2rtfdl8 DNA. After digestion with DNase, the labeled RNA was purified by phenol extraction and its hybridization with DNA fixed on a nitrocellulose membrane filter was measured according to the method of Gillespie and Spiegelman (1965) with some modifications. The results indicated that about 3% of the total labeled RNA produced with ,~r/fal8 DNA as a template could form RNA. DNA hybrids with pTUA1 DNA, whereas no crosshybridization was detected between RSF2124 DNA and )Lrtfdl8 RNA. Furthermore, the hybridization-competition study showed that RNA formed with )~r/fdl8 DNA, but not with 2DNA, could compete effectively with the labeled RNA (data not shown). In conclusion, the above findings indicate that a gene for EF-Tu, tufA, has been cloned from 2fus3 within the colicin E1 gene. From the analysis of the pattern of Sma fragments, the orientation of the inserted fragment was deduced as opposite to colicin E1 gene in terms of its direction of transcription. pTUA1 DNA as well as its EcoRI fragments were active in supporting synthesis of EF-Tu in a cell-free system and R N A . D N A hybrid formation was observed between pTUA1 DNA and RNA transcribed from 2r/fal8 DNA. The availability of tufA DNA in substantial amounts would be useful for its sequence analysis and also for quantitation of EF-Tu mRNA synthesized in a cell-free system. Studies along this line are in progress and will be reported elsewhere (Yokota et al., manuscript in preparation; and Shibuya et al., manuscript in preparation). Acknowledgment. We thank Dr. M. Nomura, University of Wisconsin for providing the bacterial strains NO 1380 ()fus3), and NO t 736 (2r/f'~18). Thanks are also due to Dr. H. Uchida for his valuable advice.
References Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map ofEscherichia coli K-12. Bacteriol. Rev. 40, 116 167 (1976) Blnmenthal, R.M., Lemaux, P.G., Neidhardt, F.C., Dennis, P.P.:
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M. Shibuya et al. : Cloning of an EcoRI Fragment Carrying E. coli tufA Gene
The effects of the relA gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in Escherichia coli B. Mol. Gen. Genet. 149, 291 296 (1976) Cameron, J.R., Panasenko, S.M., Lehman, I.R., Davis, R.W.: In vitro construction of bacteriophage 2 carrying segments of the Escherichia coli chromosome: Selection of hybrids containing the gene for DNA ligase. Proc. Natl. Acad. Sci. U.S.A. 72, 3416-3420 (1975) Dougan, G., Saul, M., Warren, G., Sherratt, D.: A functional map of plasmid ColE1. Mol. Gen. Genet. 158, 325-327 (1978) Furano, A.V. : The elongation factor Tu coded by the tufa gene of Escherichia coli K-12 is almost identical to that coded by the tufB gene. J. Biol. Chem. 252, 2154--2157 (1977) Furano, A.V.: Direct demonstration of duplicate tufgenes in enteric bacteria. Proc. Natl. Acad. Sci. U.S.A. 75, 3104-3108 (1978) Furano, A.V., Wittel, F.P.: Syntheses of elongation factors Tu and G are under stringent control in Escherichia coli. J. Biol. Chem. 251, 898-901 (1976) Gillespie, D., Spiegelman, S. : A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12, 829 842 (1965) Gordon, J. : Regulation of the in vivo synthesis of the polypeptide chain elongation factors in Escherichia coli. Biochemistry 9, 912-917 (1970) Jaskunas, S.R., Fallon, A.M., Nomura, M., Williams, B.G., Blattner, F.R.: Expression of ribosomal protein genes cloned in Charon vector phages and identification of their promoters. J. Biol. Chem. 252, 7355-7364 (1977) Jasknnas, S.R., Lindahl, L., Nomura, M., Burgess, R.R. : Identification of two copies of the gene for the elongation factor E F-Tu in E. coli. Nature 257, 458-462 (1975) Kaziro, Y.: The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim. Biophys. Acta 505, 95-127 (1978) Kirschbaum, J.B., Konrad, E.B. : Isolation of a specialized lambda transducing bacteriophage carrying the beta subunit gene for Escherichia coli ribonucleic acid polymerase. J. Bacteriol. 116, 517-526 (1973) Lindahl, L., Post, L., Zengel, J., Gilbert, S.F., Strycharz, W.A., Nomura, M. : Mapping of ribosomal protein genes by in vitro
protein synthesis using DNA fragments of 2fus3 transducing phage DNA as templates. J. Biol. Chem. 252, 7365-7383 (1977) Miller, D.L., Nagarkatti, S., Laursen, R.A., Parker, J,, Friesen, J.D.: A comparison of the activities of the products of the two genes for elongation factor Tu. Mol. Gen. Genet. 159, 57~i2 (1978) Miller, D.L., Weissbach, H. : Factors involved in the transfer of aminoacyl-tRNA to the ribosome. In: Molecular mechanisms of protein biosynthesis (Weissbach, H., and Pestka, S., eds.), pp. 323-373. New York: Academic Press 1977 Miller, J.H. : Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory 1972 Miyajima, A., Kaziro, Y. : Coordination of level of elongation factors Tu, Ts, and G, and ribosomal protein S1 in Escherichia coli. J. Biochem. (Tokyo) 83, 453462 (1978) Nomura, M. : Some remarks on recent studies on the assembly of ribosomes. In: Nucleic acid-protein recognition (Vogel, H.J., ed.), pp. 443-467. New York: Academic Press 1977 Nomura, M., Morgan, E.A., Jaskunas, S.R. : Genetics of bacterial ribosomes. Annu. Rev. Genet. 11,297-347 (1977) Reeh, S., Pedersen, S., Friesen, J.D.: Biosynthetic regulation of individual proteins in relA + and relA strains of Escherichia coli during amino acid starvation. Mol. Gen. Genet. 149, 279-289 (1976) So, M., Gill, R., Falkow, S. : The generation of a ColE1-Ap r cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet. 142, 239549 (1975) Weber, K., Osborn, M. : The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 44064412 (1969) Zubay, G., Chambers, D.A., Cheong, L.C.: Cell-free studies on the regulation of the lae operon. In: The lactose operon (Beckwith, J.R., and Zipser, D., eds.), pp. 375 391. New York: Cold Spring Harbor Laboratory 1970
Communicated
b y T. Y u r a
Received September 28, 1978
Short Communication
Cloning of an EcoRI Fragment Carrying E. coil tufA Gene Masabumi Shibuya, Hiroko Nashimoto, and Yoshito Kaziro Institute of Medical Science, University of Tokyo, Takanawa, Minatoku, Tokyo 108, Japan
Summary. EcoRI fragments of the transducing phage 2fus3 DNA have been linked to the ColE1 derivative plasmid RSF2124 (ColE1-Ap r) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUA1 was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUA1 is based on the following observations: (1) ability of pTUA1 DNA and its EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) R N A . D N A hybridization of RNA transcribed from phage 2rlfdl 8 carrying tufB with DNA from pTUA1.
Polypeptide chain elongation factor Tu promotes a GTP-dependent binding of aminoacyl-tRNA to ribosomes. The protein has a molecular weight of 47,000, and its properties, structure and function have been extensive!y studied (for recent reviews, see Miller and Weissbach, 1977; and Kaziro, 1978). It is present in E. coli cells relatively in a large quantity (Gordon, 1970) and its level under balanced growth increases as cells grow more rapidly (Furano and Wittel, 1976; Reeh et al., 1976; Blumenthal et al., 1976; Miyajima and Kaziro, 1978), The genetic element for EF-Tu is located in two distinct loci on the E. coli chromosome (Jaskunas et al., 1975); one at 72min (tufA) and the other at 88 rain (tufB) on a recalibrated map of E. coli (Bachmann et al., 1976). The transducing phages containing tufA and tufB have been isolated and their fine genetic organizations have been established (Nomura, Morgan and Jaskunas, 1977). In this paper, we report cloning of the tufA gene from 2fus3 DNA into ColE1 derivative plasmid RSF2124 (ColE1Ap r) creating a new hybrid plasmid designated pTUA1. Some properties of the cloned DNA are also described. For offprints contact: M. Shibuya
2fus3 DNA was prepared according to Miller (1972) from phage 2cI857S7fus3 produced by the heat-induced culture of NO1380 (2K401-3RrecA (2cI857S7xis665156519) (2fus3)). The DNA was dialyzed against, and stored in, 10 mM Tris-HCl (pH 7.5) containing 10 mM MgC12, 100 mM NaC1 and 10 mM dithiothreitol (EcoRI buffer). RSF2124 DNA was isolated from the cleared lysate of TM201 (E. coli K12, thy, trp, str~(RSF2124) (So, Gill, and Falkow, 1975)) by phenol extraction, Bio-Rad A5m gel filtration, and further purified by two cycles of ethidium bromide-CsC1 density equilibrium centrifugation, followed by dialysis against EcoRI buffer. For construction of the recombinant DNA molecule of RSF2124 containing an EcoRI fragment carrying tufA, a mixture of 4 ~g of RSF2124 DNA and 8 gg of 2fus3 DNA was digested by EcoRI endonuclease at 37° C. After heating for 10rain at 70° C, 0.1 mM ATP (final concentration), and an excess of T4 ligase were added to the mixture, and the incubation was further continued for 4 h at 10° C. After heating again for 10 rain at 70° C, the reaction mixture was directly used to transform cells of E. coli C600 (thr, leu, thi, lac) grown in PC medium (Cameron et al., 1975). Ampicilin-resistant transformants which did not produce colicin were selected. Since tufA is located on the 8.5% EcoRI fragment of transducing phage )fus3 DNA (Jaskunas et al., 1977; Lindahl et al., 1977), we surveyed the plasmid DNA of about 50 transformants obtained as above, and found that 5 transformants contained about 8.5 to 9% EcoRI fragments cloned in RSF2124. Among 5 such clones, one designated pTUAI was found to contain the 8.5% EcoRI fragment (Fig. 1A slot 1). The presence of the 8.5% EcoRI fragment from )fus3 DNA in pTUA1 DNA was further confirmed by its cleavage pattern with Sma. As shown in Fig. 1 C, the fragment possesses 4 Sma sites and the digestion of the fragment with Sma would yield 5 fragments, i.e. 0.9, 0.6, 2.1 a, 2.1 b, and 2.3%-2units. 0026-8925/79/0170/0231/$01.00
232
M. Shibuya et al. : Cloning of an EcoRI Fragment Carrying E. coli tufa Gene
A
B
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C 0.9% 0.6 Eco R 1 Sm(3 /
sm\2 .oz/ ......
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Fig. 1A-C. Agarose gel electrophoresis of endonuclease-digested pTUA1 and 2fus3 DNA and the orientation of the tufA-containing EcoRI fragment in pTUAI DNA. About 5-10 ~tg of DNA was digested with EcoRI,or Srna, and at the end of digestion, the reaction mixture was supplemented with one-sixth volume of the solution containing 60 mM Na2EDTA, 0.6% sodium dodecylsulfate (SDS), 0.6% bromphenol blue and 60% glycerol, and was applied onto a 4 ram-thick 0.8% agarose slab gel. Electrophoreses were performed in a buffer containing 40 mM Tris-acetate (pH 7.4), 20 mM sodium acetate, 2 mM NazEDTA and 0.5 ~tg/ml ethidium bromide for 5 to 6 hrs at 3 V/cm. (A) slot 1, pTUA1 DNA digested with EcoRI; and slot 2, )~fus3 DNA digested with EcoRI. The arrow indicates the position of EeoRI-digested RSF2124 DNA. (B) slot 1, pTUA1 DNA digested with Sma; and slot 2, 2fus3 DNA digested with Sma. The numbers to the right of (A) and (B) indicate the size of 2fus3 fragments in %-2units. The numbers in parentheses are fragments derived from the cohesive end of 2DNA. (C) Orientation of the 8.5% EcoRI fragment within pTUAI DNA showing the cleavage sites of endonucleases EcoRI and Sma. The alignment of Sma fragments in the 8.5 % EcoRI fragment is based on the observations of Jaskunas et al. (1977), Lindahl et al. (1977), and Furano (1978). More recently, we have proved this unequivocally by comparing the sequence of tufa DNA and that of EF-Tu protein (Yokota et al., unpublished)
Now, the analysis of Sma fragments of pTUA1 DNA revealed the presence of three distinct bands, 21.5, 4.7, and 2.1% fragments, on agarose gel electrophoresis (Fig. 1B slot 1). The 2.1% Sma fragment is characteristic to the 8.5% EcoRI fragment of 2fus2(3) (Nomura, 1977). The 0.6% Srna fragment was not detected in Fig. 1 B, but was clearly observed by 3.5% acrylamide-0.1% bisacrylamide gel electrophoresis (data not shown). Plasmid RSF2124 DNA (23%-2unit) has one Sma site in its colicin E1 gene, at about 2.4%-)mnit from the EcoRI site (Dougan et al., 1978). The appearance of 4.7 (2.3+2.4) and 21.5 (0.9+20.6)% Srna fragments in the Sma digestion pattern of pTUA1 DNA indicates that the 8.5% EcoRI fragment is oriented in pTUA1 DNA as illustrated in Fig. 1 C. To confirm that the entire structural gene for EFTu is cloned in the plasmid pTUA1, we analyzed proteins synthesized in the cell-free system under the direction of pTUA1 DNA or its fragments produced by cleavage with EcoRI or Sma restriction endonuclease. The DNA-dependent protein-synthesizing system used in the present study was similar to Zubay
et al. (1970) but was modified as follows. The $30 fraction derived from E. coli PR13 was treated with polyethylene glycol-6000 to remove endogenous DNA. The DNA-free polyethylene glycol-treated extracts were then supplemented with unwashed ribosomes. The incorporation of labeled amino acids in this system was completely dependent on the presence of added DNA. The details of the procedure will be published elsewhere. As shown in Fig. 2, when intact pTUA1 DNA was utilized as a template, the proteins formed contained several bands migrating close to authentic EFTu on SDS-polyacrylamide gel electrophoresis (slot 1). On digestion of the template with EcoRI, the radioactivity in the EF-Tu band was much increased to become a predominant band (slot 2). Since the natural promoter for tufA transcription is not located on the 8.5% EcoRI fragment (Jaskunas et al., 1977), the creation of some artificial initiation site by EcoRI digestion might be responsible for the increased synthesis of EF-Tu. As expected, no band corresponding to EF-Tu was seen after digestion of pTUA1 DNA with Sma (slot 3) and also in the control
M. Shibuya et al. : Cloning of an EcoRI Fragment Carrying E. coli tufA Gene
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Fig. 2. SDS-polyacrylamide gel electrophoresis of proteins synthesized in the cell-free system using intact or restriction endonuclease digested pTUA1 DNA as a template. Proteins were synthesized in a cell-free system prepared as described in the text. The reaction mixture contained in a final volume of 0.1 ml; 40 mM Tris-acetate (pH 8.2), 60 mM potassium acetate, 30 mM NHgC1, 15 mM magnesium acetate, 1.5 mM dithiothreitol, 3% (w/v) polyethylene glycol-6000, 0.5 mM each CTP, UTP, and GTP, 2 mM ATP; 0.7 gg ofpyruvate kinase, 20 mM phosphoenolpyruvate, 6 gg of folinic acid; 40 gM of [14C]leucine (340 I,tCi/~tmol), 15 gM of [14C]phenylalanine (420 gCi/gmol), and 0.2 mM each of other 18 amino acids; 120 gg of polyethylene glycol-treated $30 proteins, 2 Az60 units of unwashed ribosomes; 20 gg of uncharged tRNA, and 4 gg of DNA used as a template. The components were mixed at 0°C and incubated for 60min at 37* C. An aliquot (15 gl) was taken from each reaction mixture, and the radioactive proteins formed was electrophoresed on the slab gels containing 7.5% acrylamide - 0.2% bisacrylamide and 0.1% SDS (Weber and Osborn, 1969). The templates used are: (1) intact pTUA1 DNA, (2) EcoRIdigested pTUA1 DNA, (3) Sma-digested pTUA1 DNA, (4) intact RSF2124 DNA, (5) EcoRI-digested RSF2124 DNA, and (6) Smadigested RSF2124 DNA. Position of EF-Tu was indicated by an arrow. The amount of EF-Tu synthesized in each sample was roughly estimated using anti-EF-Tu antibody and was shown in the bottom of each slot using a symbol of ( - ) , nondetectable; (+), detectable; and (+ + +), abundant, Anti-EF-Tu immunoglobulin was a gift from Mr. A. Miyajima of this laboratory
system directed by either an intact (slot 4) or EcoRItreated (slot 5) vehicle RSF2124 DNA. For further confirmation of pTUA1 DNA-directed synthesis of EF-Tu, specific immunoprecipitation with anti-EF-Tu antibody and gel electrophoresis of the immune-precipitates were carried out according to Miyajima and Kaziro (1978). The results clearly indicated that EF-Tu was synthesized in the presence of pTUA1 DNA or its EcoRI digested fragments, and the amount of EF-Tu synthesized was severalfold higher in the latter as compared to the former (data not shown).
233
It has been shown by Jaskunas et al. (1975) that another gene for EF-Tu, i.e. tufB, is encoded in transducing phage 2r/fal8 (Kirschbaum and Konrad, 1973). Recent evidence indicates that the proteins produced by tufa and tufB are very similar, if not identical, in terms of their molecular weight, isoelectric point, pattern of tryptic digestion, and catalytic properties (Jaskunas et al., 1975; Furano, 1977; Miller et al., 1978). Therefore, it is expected that tufB DNA as well as its in vitro RNA transcripts would form a hybrid with pTUA1 DNA. To test this possibility, we have synthesized 3H-labeled RNA using E. coli RNA polymerase holoenzyme under the direction of 2rtfdl8 DNA. After digestion with DNase, the labeled RNA was purified by phenol extraction and its hybridization with DNA fixed on a nitrocellulose membrane filter was measured according to the method of Gillespie and Spiegelman (1965) with some modifications. The results indicated that about 3% of the total labeled RNA produced with ,~r/fal8 DNA as a template could form RNA. DNA hybrids with pTUA1 DNA, whereas no crosshybridization was detected between RSF2124 DNA and )Lrtfdl8 RNA. Furthermore, the hybridization-competition study showed that RNA formed with )~r/fdl8 DNA, but not with 2DNA, could compete effectively with the labeled RNA (data not shown). In conclusion, the above findings indicate that a gene for EF-Tu, tufA, has been cloned from 2fus3 within the colicin E1 gene. From the analysis of the pattern of Sma fragments, the orientation of the inserted fragment was deduced as opposite to colicin E1 gene in terms of its direction of transcription. pTUA1 DNA as well as its EcoRI fragments were active in supporting synthesis of EF-Tu in a cell-free system and R N A . D N A hybrid formation was observed between pTUA1 DNA and RNA transcribed from 2r/fal8 DNA. The availability of tufA DNA in substantial amounts would be useful for its sequence analysis and also for quantitation of EF-Tu mRNA synthesized in a cell-free system. Studies along this line are in progress and will be reported elsewhere (Yokota et al., manuscript in preparation; and Shibuya et al., manuscript in preparation). Acknowledgment. We thank Dr. M. Nomura, University of Wisconsin for providing the bacterial strains NO 1380 ()fus3), and NO t 736 (2r/f'~18). Thanks are also due to Dr. H. Uchida for his valuable advice.
References Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map ofEscherichia coli K-12. Bacteriol. Rev. 40, 116 167 (1976) Blnmenthal, R.M., Lemaux, P.G., Neidhardt, F.C., Dennis, P.P.:
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M. Shibuya et al. : Cloning of an EcoRI Fragment Carrying E. coli tufA Gene
The effects of the relA gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in Escherichia coli B. Mol. Gen. Genet. 149, 291 296 (1976) Cameron, J.R., Panasenko, S.M., Lehman, I.R., Davis, R.W.: In vitro construction of bacteriophage 2 carrying segments of the Escherichia coli chromosome: Selection of hybrids containing the gene for DNA ligase. Proc. Natl. Acad. Sci. U.S.A. 72, 3416-3420 (1975) Dougan, G., Saul, M., Warren, G., Sherratt, D.: A functional map of plasmid ColE1. Mol. Gen. Genet. 158, 325-327 (1978) Furano, A.V. : The elongation factor Tu coded by the tufa gene of Escherichia coli K-12 is almost identical to that coded by the tufB gene. J. Biol. Chem. 252, 2154--2157 (1977) Furano, A.V.: Direct demonstration of duplicate tufgenes in enteric bacteria. Proc. Natl. Acad. Sci. U.S.A. 75, 3104-3108 (1978) Furano, A.V., Wittel, F.P.: Syntheses of elongation factors Tu and G are under stringent control in Escherichia coli. J. Biol. Chem. 251, 898-901 (1976) Gillespie, D., Spiegelman, S. : A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12, 829 842 (1965) Gordon, J. : Regulation of the in vivo synthesis of the polypeptide chain elongation factors in Escherichia coli. Biochemistry 9, 912-917 (1970) Jaskunas, S.R., Fallon, A.M., Nomura, M., Williams, B.G., Blattner, F.R.: Expression of ribosomal protein genes cloned in Charon vector phages and identification of their promoters. J. Biol. Chem. 252, 7355-7364 (1977) Jasknnas, S.R., Lindahl, L., Nomura, M., Burgess, R.R. : Identification of two copies of the gene for the elongation factor E F-Tu in E. coli. Nature 257, 458-462 (1975) Kaziro, Y.: The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim. Biophys. Acta 505, 95-127 (1978) Kirschbaum, J.B., Konrad, E.B. : Isolation of a specialized lambda transducing bacteriophage carrying the beta subunit gene for Escherichia coli ribonucleic acid polymerase. J. Bacteriol. 116, 517-526 (1973) Lindahl, L., Post, L., Zengel, J., Gilbert, S.F., Strycharz, W.A., Nomura, M. : Mapping of ribosomal protein genes by in vitro
protein synthesis using DNA fragments of 2fus3 transducing phage DNA as templates. J. Biol. Chem. 252, 7365-7383 (1977) Miller, D.L., Nagarkatti, S., Laursen, R.A., Parker, J,, Friesen, J.D.: A comparison of the activities of the products of the two genes for elongation factor Tu. Mol. Gen. Genet. 159, 57~i2 (1978) Miller, D.L., Weissbach, H. : Factors involved in the transfer of aminoacyl-tRNA to the ribosome. In: Molecular mechanisms of protein biosynthesis (Weissbach, H., and Pestka, S., eds.), pp. 323-373. New York: Academic Press 1977 Miller, J.H. : Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory 1972 Miyajima, A., Kaziro, Y. : Coordination of level of elongation factors Tu, Ts, and G, and ribosomal protein S1 in Escherichia coli. J. Biochem. (Tokyo) 83, 453462 (1978) Nomura, M. : Some remarks on recent studies on the assembly of ribosomes. In: Nucleic acid-protein recognition (Vogel, H.J., ed.), pp. 443-467. New York: Academic Press 1977 Nomura, M., Morgan, E.A., Jaskunas, S.R. : Genetics of bacterial ribosomes. Annu. Rev. Genet. 11,297-347 (1977) Reeh, S., Pedersen, S., Friesen, J.D.: Biosynthetic regulation of individual proteins in relA + and relA strains of Escherichia coli during amino acid starvation. Mol. Gen. Genet. 149, 279-289 (1976) So, M., Gill, R., Falkow, S. : The generation of a ColE1-Ap r cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet. 142, 239549 (1975) Weber, K., Osborn, M. : The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 44064412 (1969) Zubay, G., Chambers, D.A., Cheong, L.C.: Cell-free studies on the regulation of the lae operon. In: The lactose operon (Beckwith, J.R., and Zipser, D., eds.), pp. 375 391. New York: Cold Spring Harbor Laboratory 1970
Communicated
b y T. Y u r a
Received September 28, 1978
