(Received for publication, March 13, 1995; and in revised form, May 12, 1995)
From the
The sequences of the cDNAs for the mitochondrial translational
elongation factor Ts (EF-Ts
In Escherichia coli, elongation factor Tu (EF-Tu)
The stability of the 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
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.).
Figure 1:
Strategy used to isolate partial cDNA
clones of EF-Ts
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
Figure 2:
Primary sequences of bovine and human
liver EF-Ts
The mature form of bovine EF-Ts
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.
Figure 4:
Southern analysis for the determination of
the number of copies of genes for EF-Ts
Figure 5:
Multiple tissue Northern blot analysis of
the EF-Ts
Figure 6:
SDS-PAGE and Western blot analysis of the
expression of EF-Ts
When the mature form of
EF-Ts
The ability of the E. coli EF-Tu
Figure 7:
Activity of the E. coli EF-Tu
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].
) 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.
(
)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-Tu
GDP is released from the ribosome. Elongation factor Ts
(EF-Ts) catalyzes the nucleotide exchange reaction promoting the
formation of EF-Tu
GTP from EF-Tu
GDP(2) . The
guanine nucleotide exchange reaction occurs through the formation of an
intermediate EF-Tu
Ts 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-Tu
EF-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-Tu
GDP complex is thought to
exchange directly with GTP present in the
(EF-Tu
EF-Ts
GTP)
dimer. Mammalian mitochondrial
EF-Tu and EF-Ts (EF-Tu
and EF-Ts
) have been
purified as a tightly associated complex (EF-Tu
Ts
)
from bovine liver(5, 6) . The EF-Tu
Ts
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.
EF-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.
-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.
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
Total RNA was extracted from bovine liver by
the guanidinium thiocyanate procedure(9) . Poly(A)-specific cDNA and PCR
Amplification
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.
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 NH
Cl, 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-Tu
EF-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-Tu
EF-Ts
were produced by Pel-Freez Biologicals
(Rogers, AK).
(
)Western blotting was done by
using the enhanced chemiluminescent detection system of Amersham Corp.
Cloning of Bovine and Human Liver EF-Ts
EF-TscDNAs
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-Tu
Ts
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
.
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.
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.
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.
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.
Analysis of the EF-Ts
For determination of the number of copies
of the gene encoding bovine liver EF-TsGenes
, 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.
(
)
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-Ts
There are no reports on the
expression of EF-TsmRNA in
Different Human Tissues
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.
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-Ts
Both the precursor of
bovine liver EF-Tsin E. coli
(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-Tu
Ts
. 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-Tu
Ts
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.
. 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-Tu
Ts
. Lanes1 and 6, no IPTG induction; lanes2 and 7, the mature form of EF-Ts
purified under denaturing conditions; lanes3 and 8, EF-Tu
Ts
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).
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) .
EF-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-Tu
Ts
complex from bovine liver.
The E. coli EF-Tu
EF-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.
EF-Ts
complex and bovine liver
EF-Tu
EF-Ts
in poly(U)-directed polymerization. The
indicated amounts of EF-Tu
Ts
purified from bovine
liver (
) or of the E. coli EF-Tu
Ts
complex (
) were tested for activity as described under
``Experimental Procedures.''
, 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.