©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of Mitochondrial Translational Elongation Factor Ts from Bovine and Human Liver (*)

(Received for publication, March 13, 1995; and in revised form, May 12, 1995)

Hong Xin (1), Velinda Woriax (1), William Burkhart (3), Linda L. Spremulli (1) (2)(§)

From the  (1)Department of Chemistry and the (2)Lineberger Comprehensive Cancer Research Center, University of North Carolina, Chapel Hill, North Carolina 27599-3290 and the (3)Department of Bioanalytical and Structural Chemistry, Glaxo Research Institute, Research Triangle Park, North Carolina 27709

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The sequences of the cDNAs for the mitochondrial translational elongation factor Ts (EF-Ts) from bovine and human liver have been obtained. The deduced amino acid sequence of bovine liver EF-Ts is 338 residues in length and includes a 55-amino acid signal peptide and a mature protein of 283 residues. The sequence of the mature form of bovine EF-Ts is 91% identical to that of human EF-Ts and 29% identical to Escherichia coli EF-Ts. Southern analysis indicates that there are two genes for EF-Ts in bovine liver chromosomal DNA. A 224-base pair intron is located near the 5`-end of at least one of these genes. Northern analysis using a human multiple tissue blot indicates that EF-Ts is expressed in all tissues, with the highest levels of expression in skeletal muscle, liver, and kidney. Both the mature and precursor forms of bovine liver EF-Ts have been expressed in E. coli as histidine-tagged proteins. The mature form of EF-Ts forms a complex with E. coli elongation factor Tu. This complex is active in poly(U)-directed polymerization of phenylalanine. The precursor form is expressed as a 42-kDa protein, which is rapidly degraded in the cell.


INTRODUCTION

In Escherichia coli, elongation factor Tu (EF-Tu)()facilitates the binding of aminoacyl-tRNA to the ribosome during the elongation cycle of protein biosynthesis(1) . Following A-site binding of the correct aminoacyl-tRNA, EF-Tu catalyzes the hydrolysis of GTP, and EF-TuGDP is released from the ribosome. Elongation factor Ts (EF-Ts) catalyzes the nucleotide exchange reaction promoting the formation of EF-TuGTP from EF-TuGDP(2) . The guanine nucleotide exchange reaction occurs through the formation of an intermediate EF-TuTs complex(3) . In contrast to E. coli, during the elongation cycle of protein synthesis in Thermus thermophilus, a dimeric form of EF-Ts binds two molecules of EF-Tu, forming an (EF-TuEF-Ts) structure, which is extremely stable and cannot be dissociated in the absence of protein-denaturing reagents(4) . In this organism, GDP present in the EF-TuGDP complex is thought to exchange directly with GTP present in the (EF-TuEF-TsGTP) dimer. Mammalian mitochondrial EF-Tu and EF-Ts (EF-Tu and EF-Ts) have been purified as a tightly associated complex (EF-TuTs) from bovine liver(5, 6) . The EF-TuTs complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. In this respect, the mitochondrial factors differ significantly from the corresponding E. coli factors and show some resemblance to thermophilic EF-Tu and EF-Ts.

The stability of the EF-TuEF-Ts complex is thought to be determined largely by the nature of the EF-Ts component. For example, EF-Ts from thermophilic bacteria forms strong complexes with E. coli EF-Tu, whereas E. coli EF-Ts produces only weak complexes with thermophilic EF-Tu(7) . In addition, chloroplast EF-Ts from Euglena gracilis forms a tighter complex with E. coli and chloroplast EF-Tu than does the E. coli factor (8) . It is not clear what features of EF-Ts modulate the strength of its interaction with EF-Tu.

The genes for EF-Ts from several prokaryotes have been cloned and sequenced, as has the gene for chloroplast EF-Ts from the thermophilic red algae Galdieria sulphuraria. In general, the overall sequence for EF-Ts is far less conserved than the sequence for EF-Tu, but there is some conservation located in the NH-terminal one-third of the protein. In this work, cDNAs encoding EF-Ts have been cloned and sequenced from both bovine and human liver. In addition, bovine liver EF-Ts has been expressed in E. coli.


EXPERIMENTAL PROCEDURES

Peptide Sequence Analysis of Bovine Liver EF-Ts

EF-TuTs was purified as described(5) . The EF-Tu and EF-Ts components were separated by reverse-phase high performance liquid chromatography using a Brownlee RP300 column (2.1 100 mm) with a linear gradient of acetonitrile, 0.1% trifluoroacetic acid (20-64%) over 60 min. The fraction identified as EF-Ts was dried, dissolved in 8 M urea, and incubated at 50 °C for 30 min. The solution was diluted to 4 M urea with 0.2 M Tris-HCl, pH 8.5. Sequence-grade endoproteinase Lys-C was added (5 µg), and the sample was incubated at 37 °C for 20 h. The resultant peptides were separated on a Brownlee RP300 column (1.0 250 mm) with a linear gradient of acetonitrile, 0.1% trifluoroacetic acid (8-64%) over 90 min. The prominent peaks were sequenced on an Applied Biosystems 477A liquid-pulse sequencer connected to an Applied Biosystems 120A phenylthiohydantoin analyzer.

Synthesis of EF-Ts-specific cDNA and PCR Amplification

Total RNA was extracted from bovine liver by the guanidinium thiocyanate procedure(9) . Poly(A) RNA was purified by oligo(dT)-cellulose chromatography(10) . Single-stranded cDNAs were synthesized by reverse transcription of 5 µg of mRNA using primer 3 or 4 (see Table 1) or primer NS (GGAATTCCCTGCCTGTTTGAGATCCCCGC; the underlined sequence represents an EcoRI adaptor). Bovine liver chromosomal DNA was prepared as described(11) .



PCR amplification reaction mixtures (100 µl) contained 0.2 mM dNTPs, 2.5 units of Taq DNA polymerase, 50 pmol of the appropriate primers, the buffer system purchased with Taq DNA polymerase (Promega), and either 1 µg of bovine liver chromosomal DNA or an aliquot of the specifically primed cDNA. When the cDNA was used as template, the first five cycles were done at 94 °C for 1 min (denaturation), 56 °C for 1.5 min (annealing), and 72 °C for 2 min (polymerization). For the remaining 35 cycles, annealing was carried out at 61 °C. When chromosomal DNA was used as template, primer annealing was done at 50 °C during the first five cycles and at 55 °C in the 35 remaining cycles. In the last cycle, the reaction time at 72 °C was extended to 5 min to allow completion of chains.

Nested PCRs involving a second or third round of amplification were carried out as described above, except that they contained 1 µl of the reaction mixture obtained from the previous round of PCR as the template. The reaction mixtures were analyzed on 1.5% agarose or 3% NuSieve GTG-agarose gels. Specific bands were identified by ethidium bromide staining and eluted from the gel using a Geneclean or Mermaid kit (BIO 101, Inc.).

Screening cDNA Libraries

Approximately 5 10 plaques from two bovine liver libraries (Stratagene) and 2 10 plaques from a human liver cDNA library (CLONTECH) were screened by hybridization with a bovine liver EF-Ts cDNA probe obtained by PCR amplification and labeled using random priming (12) . Hybridizations with bovine liver libraries were carried out at 65 °C, while hybridization with the human library was done at 55 °C. Positive plaques were replated until purified, and the pBluescript SK(-) phagemid clones were excised according to the manufacturer's instructions (Stratagene).

5`-Rapid Amplification of cDNA Ends (5`-RACE)

Single-stranded cDNA was synthesized as described above using the reverse primer NS. A poly(dA) tail was added to this cDNA by terminal transferase. This cDNA was used as template for 5`-RACE-PCR (13) . A specific PCR product, 260 bp, was obtained, and two BamHI fragments from it were cloned into pTZ18R.

DNA Sequencing

EF-Ts clones were sequenced by the dideoxynucleotide chain termination method (14) and subjected to autosequencing in the University of North Carolina DNA Sequencing Facility. All clones were sequenced completely in both directions. Analysis of the sequence was done with Genetics Computer Group sequence analysis programs running on a VAX computer. Single-stranded 5`-RACE products were sequenced after two rounds of nested PCR. The single-stranded DNA was generated in the second round of PCR by using one primer in a 50-fold molar excess over the other primer.

Northern and Southern Analyses

Northern analysis of poly(A) RNA was performed using 1% agarose gels run in the presence of 1 M glyoxal and 50% dimethyl sulfoxide as described(15) . Bovine chromosomal DNA (30 µg) was digested with an optimal amount of the indicated restriction enzyme. Digests were run on 1.5% agarose gels at 60 V for 19 h. Nucleic acids were transferred to Zeta-Probe blotting membranes as recommended by the manufacturer (Bio-Rad), and blots were probed as indicated.

Expression and Purification of Mature and Precursor Forms of Bovine Liver EF-Ts

PCR was used to add an NdeI cutting site to the 5`-end and an XhoI cutting site to the 3`-end of the bovine liver cDNA encoding the precursor form of EF-Ts and to the portion of the cDNA encoding the mature form of EF-Ts. These cDNAs were then cloned into pET24c(+). E. coli BL21(DE3) was used as the host for expression. Purification of the mature and precursor forms under denaturing conditions was performed using nickel-nitrilo-triacetic acid (Ni-NTA) affinity chromatography as described by QIAGEN Inc. For the large-scale purification of the mature and precursor forms under native conditions, expression was induced by exposure of cells (1.0-1.2 A units/ml) to 0.025 mM IPTG. Cells were collected after 1 h of induction and lysed by grinding with two times the cell weight of alumina for 20 min on ice. The cell paste was resuspended in 4 volumes of buffer containing 50 mM Tris-HCl, pH 7.6, 60 mM KCl, 7 mM MgCl, 7 mM -mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 10% glycerol. Alumina was removed by centrifugation at 11,000 g at 4 °C for 15 min. The supernatant was collected and incubated with DNase I (5 µg/ml) for 15 min on ice. The extract was then subjected to centrifugation at 15,000 g for 30 min. Ni-NTA resin (0.4 ml of a 50% slurry) equilibrated in the same buffer was added to the extract for each 1 liter of original culture. This slurry was shaken at 4 °C for 1 h. The Ni-NTA resin was collected by centrifugation at 15,000 g for 10 min, rinsed with 35 ml of wash buffer (50 mM Tris-HCl, pH 7.6, 1 M NHCl, 5 mM -mercaptoethanol, 10 mM imidazole, and 10% glycerol), poured into a small column, and washed with an additional 60 ml of wash buffer. Protein was eluted from the resin using three aliquots (1 ml each) of elution buffer (50 mM Tris-HCl, pH 7.6, 40 mM KCl, 5 mM -mercaptoethanol, 0.15 M imidazole, and 10% glycerol). The eluted protein was dialyzed immediately against a 100-fold excess of buffer containing 20 mM Hepes/KOH, pH 7.0, 40 mM KCl, 1 mM MgCl, 0.1 mM EDTA, and 10% glycerol.

Assays and Western Analysis

The protein concentrations were determined by the Micro-Bradford method (Bio-Rad). The activity of the complex containing E. coli EF-Tu and EF-Ts (E. coli EF-TuEF-Ts) was measured by its ability to catalyze the poly(U)-directed polymerization of phenylalanine on E. coli ribosomes(5, 6) . One unit is defined as the incorporation of 1 pmol of [C]Phe into polypeptide at 37 °C using a 30-min incubation. Polyclonal antibodies against EF-TuEF-Ts were produced by Pel-Freez Biologicals (Rogers, AK).()Western blotting was done by using the enhanced chemiluminescent detection system of Amersham Corp.


RESULTS AND DISCUSSION

Cloning of Bovine and Human Liver EF-TscDNAs

EF-Ts is the product of a nuclear gene in mammals. To obtain cDNA clones of this factor, it was necessary to obtain partial peptide sequence information. The EF-TuTs complex was dissociated, and the two factors were separated by reverse-phase high performance liquid chromatography. EF-Ts was subjected to NH-terminal Edman degradation and to internal peptide sequence analysis following digestion with endoproteinase Lys-C. The sequences of six peptides ranging in size from 9 to 26 residues including the NH-terminal peptide were obtained (Table 1). Several of the peptides have only a low sequence identity to the sequences for the corresponding prokaryotic factors, and except for the NH-terminal peptide, their relative positions could not be predicted. Degenerate oligonucleotide primers were designed from these sequences. Forward primers Np-1 and 1 were derived from the NH-terminal peptide, and their relative positions with respect to each other were known exactly (Fig. 1). Reverse primers 3 and 4 were predicted to be located either in the middle or the COOH-terminal region of EF-Ts. The positions of primers 3 and 4 relative to each other could not be predicted. Hence, two groups of nested reverse transcriptase-PCRs were carried out (Fig. 1). Two specific cDNAs were synthesized, one using primer 3 (cDNA3) and the other using primer 4 (cDNA4). In the first round of PCR, the primer used for cDNA synthesis was used in combination with primer Np-1 derived from the NH-terminal sequence. No specific bands were visible after the first round of PCR in either group. A second round of PCR was performed using the product of cDNA3 and primers Np-1 and 4. No specific bands could be observed after this second round of PCR, suggesting that primer 3 lies to the 5`-side of primer 4. In contrast, a very strong band of 650 bp could be observed after the second round of PCR using the product of the first round of PCR derived from cDNA4 with primers 3 and Np-1 (Fig. 1). This observation confirms the idea that primer 3 lies to the 5`-side of primer 4, allowing the nested PCR to succeed. To further confirm the identity of the 650-bp product, this fragment was amplified using primers 3 and primer 1. This reaction gave a product of 600 bp, 60 bp shorter than the starting DNA. This size difference corresponds to the distance between primers Np-1 and 1. The 650-bp product was cloned into vector pTZ18R and sequenced. The deduced amino acid sequence of this fragment contained five of the peptides obtained by sequence analysis, confirming that this fragment is indeed a partial cDNA coding for bovine liver EF-Ts.


Figure 1: Strategy used to isolate partial cDNA clones of EF-Ts by nested reverse transcriptase-PCR.



Bovine liver Zap II and MAX-1 cDNA libraries were screened for additional portions of the EF-Ts cDNA. Three positive plaques were isolated from the Zap II cDNA library, and the pBluescript plasmids carrying the cDNA inserts of interest were excised in vivo. DNA sequence analysis indicated that these clones encompassed the entire 3`-region of the EF-Ts cDNA, including the poly(A) tail. Five clones were isolated in a similar manner from the MAX-1 cDNA library. They contained inserts of 135 bp from the middle of the EF-Ts coding sequence. None of eight clones isolated contained the 5`-region of the bovine liver EF-Ts cDNA, and all of the inserts had the same 5`-end. Subsequent analysis of the sequence of the EF-Ts cDNA indicated that there is a G:C-rich region just upstream of this position. Presumably, secondary structure in the mRNA resulted in a strong stop for reverse transcriptase during cDNA synthesis. To obtain the sequences from the 5`-end of the EF-Ts cDNA, 5`-RACE-PCR was carried out(13) . This approach allowed the cloning of an additional 176 bp from the 5`-end of the EF-Ts cDNA.

Analysis of the cDNA clones described above provided 1297 bp of sequence including the poly(A) tail and encompassing the entire coding region (Fig. 2). The size of the cDNA obtained corresponds well to the size estimated for the mRNA by Northern analysis (data not shown). The long open reading frame codes for the entire EF-Ts polypeptide, including a 55-amino acid mitochondrial localization signal and a mature protein of 283 amino acids. The cDNA sequence indicates that bovine liver EF-Ts has a very short 5`-untranslated region (18 bp long). Although few eukaryotic cytoplasmic mRNAs have leader regions as short as 18 nucleotides, work by Kozak (16, 17, 18) indicates that a leader of this length is generally sufficient to allow initiation. It is possible that all of the clones obtained by 5`-RACE-PCR are shorter than the actual mRNA, reflecting a strong barrier to reverse transcriptase at this position. However, 27 clones obtained by 5`-RACE-PCR all terminated within this region. The initiation codon (designated position +1) is preceded by an A residue at position -3 and followed by a T residue at position +4. Analysis of numerous translational start sites indicates that the consensus sequence has a purine at position -3 and a G residue at position +4(19) . The 3`-untranslated region is 190 bp in length and contains a polyadenylation signal (AAUAAA) 16 nucleotides before the poly(A) tail. An analysis of the encoded amino acid sequence is provided below.


Figure 2: Primary sequences of bovine and human liver EF-Ts cDNAs. A, the nucleotide sequence of the bovine liver EF-Ts cDNA is shown along with the amino acid sequence of EF-Ts beginning with the first ATG codon. Underlined sequences indicate peptides obtained from protein sequencing. *S is the NH terminus of the mature form of bovine liver EF-Ts. The doubleexclamation points indicate the position of the intron in the bovine liver EF-Ts gene. The amino acid residues under the sequence for bovine liver EF-Ts indicate the differences in this sequence from human liver EF-Ts. The human liver cDNA lacks sequence information from the 5`-end of the mRNA including a portion of the signal peptide and begins with the alanine designated **A. The mature form of human liver EF-Ts probably begins at the same position as the bovine liver EF-Ts. B, shown is the sequence of the intron in the bovine liver EF-Ts genes. This intron is located between positions 99 and 100 of the cDNA.



The bovine liver cDNA was used as a probe to screen a human liver Zap II library. Two of the 10 positive clones obtained had inserts of 1 kilobases, including a poly(A) tail. These two clones included the entire coding region for the mature form of bovine liver EF-Ts and a portion of the mitochondrial import signal (Fig. 2).

Sequence Analysis of Mammalian EF-Ts

NH-terminal sequence analysis indicates that the mature form of bovine liver EF-Ts begins with Ser-56 in the long open reading frame (Fig. 2). The mitochondrial import signal for EF-Ts is thus 55 residues long. Mitochondrial import sequences are not highly conserved in primary sequence. However, they generally lack acidic residues, are enriched in basic and hydroxylated amino acids, and can form an amphiphilic -helix or -sheet. The transit peptide for bovine EF-Ts lacks acidic residues, but is not particularly enriched in either basic or hydroxylated residues. It does not appear to be able to form an amphiphilic -helix or -sheet. At least two different pathways are believed to be involved in the processing of proteins imported into mitochondria(20) . One pathway uses a single mitochondrial processing peptidase that recognizes Arg at position -2 relative to the processing site(20) . The precursor for bovine liver EF-Ts does not appear to fit into this group. A second pathway involves sequential cleavage by two proteases. Proteins processed by this pathway generally have Arg at position -10, a hydrophobic residue at position -8, and Gly, Ser, or Thr at position -5 relative to the cut site. The precursor for bovine liver EF-Ts has His at position -10, Phe at position -8, and Gly at position -5 and may possibly be processed by this two-step pathway.

The mature form of bovine EF-Ts is 283 amino acids in length (Fig. 2) and has a molecular mass of 30,739 Da. The mature form of human liver EF-Ts also appears to have 283 residues. The two mammalian EF-Ts sequences are 91% identical (Table 2). The sequence of EF-Ts from three prokaryotes (E. coli, Spiroplasma citri, and Spirulina platensis), one chloroplast EF-Ts sequence (G. sulphuraria), and several eukaryotic EF-1 sequences have been reported(21, 22, 23, 24, 25) . The - and -subunits of the cytoplasmic factor have both been reported to function as nucleotide exchange factors(24, 25) . The bacterial factors are 250-300 residues in length, similar to the size of the mammalian mitochondrial factors. The one known gene for chloroplast EF-Ts encodes a protein of 199 residues, although it is unclear whether this gene actually encodes a functional product(23) . The only chloroplast EF-Ts studied at the protein level to date is from E. gracilis(26) and appears to be a monomer of 62 kDa, considerably larger that any of the corresponding factors known. The - and -subunits of the cytoplasmic factor EF-1 are 227 and 265 residues in length, respectively(24, 25) . A comparison of the sequences of these nucleotide exchange factors (Table 2) indicates that mammalian EF-Ts is 27-35% identical to the corresponding prokaryotic and chloroplast factors, but <21% identical to either the - or -subunit of EF-1.



The alignment of the sequences of EF-Ts from prokaryotes, chloroplasts, and mammalian mitochondria (Fig. 3) indicates that conserved regions are clustered in the NH-terminal one-third of the protein. The complete conservation of 22 residues in these factors is observed, with 20 of these amino acids being located within the first 90 residues. The longest stretch of completely conserved residues is 5 amino acids long. There is also a conserved stretch of 10 residues present if conservative substitutions are taken into account. Little information is available on the regions of EF-Ts that are important for the nucleotide exchange activity of this factor. The sequence alignment presented in Fig. 3and the observation that EF-Ts will promote GDP exchange with E. coli EF-Tu suggest that the NH-terminal one-third of the protein may play a particularly important role in this process.


Figure 3: Comparison of the amino acid sequences of EF-Ts from different organisms. The alignment of the sequences was carried out by using the PILEUP program in the Genetics Computer Group software package. See Footnote a in Table 2for definition of the designations used.



Analysis of the EF-TsGenes

For determination of the number of copies of the gene encoding bovine liver EF-Ts, a Southern blot of total DNA digested with EcoRI, BamHI, HindIII, and BglII, respectively, was probed using nucleotides 177-1182 as a probe (Fig. 4). This region was selected as a probe because PCR analysis of genomic DNA and the cDNA indicated that no introns were present in this region of the gene (data not shown). EcoRI cuts the probe once, while the other three enzymes do not have a cutting site in the probe. As indicated in Fig. 4, EcoRI digestion resulted in the appearance of four bands, while BamHI, HindIII, and BglII digestion gave two bands on the Southern blot. These results suggest that two genes encoding EF-Ts exist in bovine chromosomal DNA. PCR analysis failed to amplify genomic sequences corresponding to the cDNA, suggesting that neither of the genes detected is a pseudogene (data not shown). It has recently been observed that there are also two copies of the EF-Tu gene in bovine chromosomal DNA.()


Figure 4: Southern analysis for the determination of the number of copies of genes for EF-Ts in bovine DNA. Total DNA from bovine liver was digested with EcoRI (lane1), BglII (lane2), HindIII (lane3), and BamHI (lane4), and the fragments produced were separated by agarose gel electrophoresis as described under ``Experimental Procedures.'' The probe used extended from nucleotides 177 to 1182. The relative positions of size markers are indicated (in kilobases (kb)). The weak intensity of the smaller band in lane4 is thought to be the result of partial digestion.



An examination of the EF-Ts gene for the presence of intervening sequences was carried out by PCR amplification of chromosomal DNA using different combinations of oligonucleotide primers designed from the cDNA sequence. This analysis indicated the presence of an intron near the 5`-end of the bovine EF-Ts genes. The PCR analysis used here suggests that both genes contain a similarly sized intron. However, this procedure can only detect relatively small introns, and this interpretation must be viewed with caution. This intron (224 bp) was cloned and sequenced (Fig. 2B).

Northern Analysis of the EF-TsmRNA in Different Human Tissues

There are no reports on the expression of EF-Ts in any mammalian species to date. To investigate the relative amounts of the EF-Ts transcript in different human tissues, a multiple tissue Northern blot was analyzed using the full-length cDNA of EF-Ts as a probe. As shown in Fig. 5, EF-Ts transcripts of 1200 bases could be observed in all the human tissues analyzed. Human skeletal muscle had the most abundant level of transcripts among the tissues tested, followed by liver, kidney, and heart. Placenta, brain, pancreas, and lung had substantially lower levels of the mRNA for EF-Ts. Higher levels of expression were clearly observed in specialized tissues known to have high demands for energy production. Skeletal muscle contains not only the normal 1200-base mRNA, but also a larger transcript. Lower amounts of this other transcript are observed in several other tissues. This transcript may possibly arise from use of an alternative polyadenylation site (27) or from transcription of the two separate genes or may represent a cross-hybridizing mRNA.


Figure 5: Multiple tissue Northern blot analysis of the EF-Ts mRNA transcribed in different human tissues. The multiple tissue Northern blot contained 2 µg of poly(A) mRNAs from various human tissues. The blot was hybridized to a probe encompassing residues 177-1182 of the bovine liver cDNA. Sizes of RNA markers are shown on the left (in kilobases). Lane1, heart; lane2, brain; lane3, placenta; lane4, lung; lane5, liver; lane6, skeletal muscle; lane7, kidney; lane8, pancreas.



Expression of the Mature Form and the Precursor of EF-Tsin E. coli

Both the precursor of bovine liver EF-Ts (pre-EF-Ts) and the mature form of this factor (amino acids 56-338) were cloned into a pET expression vector. Both constructs carry a 6-residue histidine tag at the COOH terminus separated from the normal COOH terminus by a Leu-Glu linker. As indicated in Fig. 6(lane1), little, if any, material corresponding to EF-Ts was detectable in extracts of uninduced cells. However, following IPTG induction, a major band with a molecular mass of 34 kDa was observed (Fig. 6, lane2). Western analysis of samples before and after induction (lanes6 and 7) indicated that the new band reacted strongly with antibodies raised against bovine EF-TuTs. This observation demonstrates that the cells are indeed expressing the bovine mitochondrial factor. The expressed EF-Ts migrated on SDS-PAGE to a position 2 kDa larger than that observed with EF-TuTs prepared from mitochondria (Fig. 6, lanes2 and 3). This observation is not surprising since there are 9 extra amino acid residues including the His tag in the expressed form of the factor. An examination of the effects of induction of EF-Ts on the growth of the host E. coli cells indicated that the cells stopped growing within 1 h after induction, suggesting that the expression of the bovine mitochondrial factor is toxic to the cells. Maximal levels of induction were observed between 1 and 2 h following addition of IPTG, and concentrations of IPTG as low as 25 µM gave maximal levels of expression.


Figure 6: SDS-PAGE and Western blot analysis of the expression of EF-Ts. Lanes 1-5, silver-stained SDS-polyacrylamide gels; lanes 6-8, Western blot of samples corresponding to lanes 1-3 using antibodies raised against bovine liver EF-TuTs. Lanes1 and 6, no IPTG induction; lanes2 and 7, the mature form of EF-Ts purified under denaturing conditions; lanes3 and 8, EF-TuTs purified from bovine liver; lane4, the mature form of EF-Ts purified under nondenaturing conditions; lane5, E. coli EF-Tu prepared as described previously(5) . The positions of protein molecular mass markers are shown on the left.



The His-tagged form of pre-EF-Ts was expressed in E. coli as a 42-kDa protein. Pre-EF-Ts could be observed primarily in extracts made under denaturing conditions and when induction was carried out for only 30 min. Longer periods of induction resulted in the degradation of pre-EF-Ts (data not shown).

When the mature form of EF-Ts was purified by Ni-NTA chromatography under nondenaturing conditions and then analyzed by SDS-PAGE (Fig. 6, lane4), a band with a molecular mass of 44 kDa copurified with EF-Ts. This band appears to have a molecular mass identical to that of E. coli EF-Tu (lane5), indicating that the mitochondrial factor will form a complex with bacterial EF-Tu. This observation is not surprising since EF-Ts can promote guanine nucleotide exchange with prokaryotic EF-Tu(5) .

The ability of the E. coli EF-TuEF-Ts complex to function in the poly(U)-directed polymerization of phenylalanine was examined. As shown in Fig. 7, this complex was active in the in vitro assay. The specific activity obtained for the heterologous complex (140,000 units/mg) is 30% of the activity that is obtained with the homologous EF-TuTs complex from bovine liver. The E. coli EF-TuEF-Ts complex functions catalytically in the polymerization assay, with 10 rounds of phenylalanine incorporated into peptide for each complex present. A more detailed analysis of the properties of this heterologous complex is now underway.


Figure 7: Activity of the E. coli EF-TuEF-Ts complex and bovine liver EF-TuEF-Ts in poly(U)-directed polymerization. The indicated amounts of EF-TuTs purified from bovine liver () or of the E. coli EF-TuTs complex () were tested for activity as described under ``Experimental Procedures.''




FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM32734. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L37935 [GenBank® Link]and L37936[GenBank® Link].

§
To whom correspondence should be addressed: Dept. of Biochemistry CB 3290, Lineberger Comprehensive Cancer Research Center, University of North Carolina, Chapel Hill, NC 27599-3290. Tel.: 919-966-1567; Fax: 919-962-2388; lls{at}uncvx1.oit.unc.edu

The abbreviations used are: EF-Tu, elongation factor Tu; EF-Ts, elongation factor Ts; EF-1, elongation factor 1; EF-Tu, mitochondrial EF-Tu; EF-Ts, mitochondrial EF-Ts; pre-EF-Ts, precursor of bovine liver EF-Ts; PCR, polymerase chain reaction; 5`-RACE, 5`-rapid amplification of cDNA ends; bp, base pair(s); IPTG, isopropyl-1-thio--D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis.

V. Woriax and L. Spremulli, manuscript in preparation.

V. Woriax, W. Burkhart, and L. Spremulli, manuscript in preparation.


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