(Received for publication, November 10, 1995)
From the
A complete cDNA clone encoding bovine mitochondrial
translational initiation factor 2 (IF-2) has been
obtained. The regions of the cDNA corresponding to mature IF-2
and several of its functional domains have been expressed in Escherichia coli as histidine-tagged proteins. The precursor
(
90 kDa) and mature (
85 kDa) forms of IF-2
are
toxic to E. coli and can only be expressed at low levels.
Shorter forms of this factor (
80 and
72 kDa) are also found
during the expression of mature IF-2
. The various forms of
IF-2
can be separated by high performance liquid
chromatography. All of these forms are active in promoting the
GTP-dependent binding of formyl-Met-tRNA to the small subunit of either E. coli or bovine mitochondrial ribosomes. IF-2
can bind to mitochondrial ribosomes in the absence of GTP,
initiator tRNA, or messenger RNA. The presence of GTP stimulates
IF-2
binding to ribosomes about 3-fold. IF-2
interacts only weakly with GTP or with the initiator tRNA in the
absence of ribosomes. Molecular dissection of IF-2
shows
that a long deletion (
150 amino acid residues) from the
NH
-terminal region does not affect its activity in
vitro. The COOH domain of IF-2
(amino acid residues
332-727) can bind to ribosomes even though it does not promote
initiator-tRNA binding.
The initiation of protein biosynthesis has been widely studied
in the prokaryotic and eukaryotic cytoplasmic systems. But the process
of translational initiation in mitochondria is poorly understood. The
only initiation factor that has been identified to date in animal
mitochondria is translational initiation factor 2 (IF-2) (
)and many questions remain about how the initiation of
protein biosynthesis occurs in this organelle(1) . IF-2
promotes the binding of the initiator tRNA to the 28 S ribosomal
subunit in the presence of GTP and a mRNA(1) . This factor
belongs to the family of GTPases which are molecular switches capable
of alternating between an active (IF-2
GTP) and an
inactive (IF-2
GDP) conformation(2) .
We
have obtained a complete cDNA clone encoding bovine
IF-2(3) . The mature form of this protein is
predicted to have 698 amino acid residues proceeded by a 29-amino acid
mitochondrial import signal at the NH
terminus. The mature
form of bovine IF-2
can be divided into three regions. The
NH
-terminal region (Leu-30-Ser-180) is rich in
charged amino acids. The function of this domain is unclear even for
prokaryotic IF-2s. The middle region (Pro-181-Asn-330)
encompasses the nucleotide binding domain (G-domain) and is quite
homologous to the G-domain of analogous factors. The similarity between
bovine IF-2
and prokaryotic IF-2s decreases in the
COOH-terminal region. Two unusual features are observed in the
COOH-terminal half of bovine IF-2
. First, the sequence
between Asp-429 and Glu-512 has 60% charged residues and has the
highest surface probability in the whole molecule. Second, there are 37
extra amino acid residues present in two clusters not found in any
prokaryotic IF-2 (3) . The DNAs encoding IF-2
from
yeast and humans have also been cloned and sequenced, although neither
of these factors has been purified and no studies on their properties
have been carried out (4, 5) .
The identification
of the important sites in IF-2 responsible for the
interaction with GTP, fMet-tRNA, and ribosomes is of particular
interest. Studies with the IF-2s from prokaryotes have led to the
identification of the GTP-binding site. However, little is known
concerning other important regions of this protein. A six-domain model
for the
form of Escherichia coli IF-2 (97 kDa) has been
proposed(6) . This model is based on data from DNA sequence
analysis, protein sequence comparisons, patterns of proteolysis, and
secondary structure predictions. The first three domains (residues
1-103, 104-155, and 156-391 of IF-2
) are in the
NH
terminus proceeding the GTP binding domain (domain IV,
residues 392-540). Domain V (residues 541-671) and VI
(residues 672-890) are defined by proteolytic cleavage patterns.
Two structurally compact and functional domains have been observed in Bacillus stearothermophilus IF-2(7) . The G-domain
corresponding to domains III-V in E. coli IF-2
(
41 kDa) contains the GTP binding site, the catalytic center for
the GTP hydrolysis, and probably also has a site that interacts with
the 50 S ribosomal subunit. The COOH-terminal domain (
24 kDa)
corresponding to domain VI in the six-domain model of E. coli IF-2
(6) probably contains the fMet-tRNA binding
site.
During the past several years, studies on structure/function
relationships in IF-2 have been hampered by the limited
amount of the native protein that can be obtained. To circumvent this
problem, the regions of the cDNA corresponding to mature IF-2
and several of its functional domains have been subcloned and
expressed in E. coli, and their properties have been
investigated.
For small scale experiments, cell cultures were harvested by centrifugation at 14,000 rpm for 1 min in a microcentrifuge. The pellets were fast frozen in a dry ice/2-propanol bath and stored at -70 °C. For large scale preparations (1-6 liters), cells were harvested by centrifugation for 20 min at 6,000 rpm in a H-6000A rotor at 4 °C. The pellets were washed in buffer JM1 and subjected to centrifugation for 15 min at 6,000 rpm in a SS-34 rotor at 4 °C. The cell pellets were then fast frozen and stored at -70 °C until use.
The level of expression following induction was evaluated by a one-step purification procedure under denaturing conditions according to the protocol provided by Qiagen, Inc. The samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5, 10, or 12% gels, and proteins present in the gel were visualized by silver staining(13) .
During purification, protein concentrations were determined
by the Bio-Rad protein assay kit using bovine serum albumin as a
standard. The NH-terminal sequence of several purified
forms of expressed IF-2
was determined using the Applied
Biosystems 477A protein sequencer with the 120A parathyroid hormone
analyzer by Mary Moyer and William Burkhart (Glaxo Research Institute,
Research Triangle Park, NC). About 100 pmol of the purified protein was
provided for protein sequencing.
Figure 1:
Molecular dissection of bovine
IF-2. The primary sequence of bovine IF-2
has
been compared with that of E. coli IF-2
. The regions of
IF-2
corresponding to the six domains of E. coli IF-2
have been labeled from II to VI. Constructs were
prepared including the precursor and mature forms of IF-2
,
the NG-, G-, GC-, and C-domains of bovine IF-2
as
indicated. The numbers indicate amino acid
residues.
The effect of the expression of
bovine IF-2 and its domains in E. coli was
determined by comparing the time courses of cell growth with and
without IPTG induction. Both the precursor and mature forms of
IF-2
are toxic to E. coli and cells stop growing
within 30 min following induction (data not shown). The expression of
the NG-, G-, GC-, and C-domains was considerably less toxic to E.
coli and cells continued to grow following induction (data not
shown). The level of expression of IF-2
or its domains
following induction was evaluated by a one-step purification procedure
on a Ni-NTA affinity resin following the preparation of cell extracts
under denaturing conditions. The effect of the time of induction on the
yield of the expressed proteins was determined. Overall, the yields of
the expressed proteins increase with time (data not shown). Low
concentrations of IPTG were found to give higher yields of mature
IF-2
(data not shown). The yield of the GC-domain of
IF-2
was about 3-4-fold higher than that of
IF-2
. Thus, the GC-domain was purified and used to prepare
antibodies to facilitate the identification of the expressed products.
These antibodies do not recognize E. coli IF-2 (data not
shown).
When the precursor of IF-2 is expressed, a
faint band designated by a small arrow with the expected molecular mass
(90 kDa) is observed on SDS-PAGE (Fig. 2A). This band
reacts strongly with antibodies raised against the GC-domain of
IF-2
(Fig. 2B, lane 1). However,
the major form seen following induction of the precursor of IF-2
migrates at 80 kDa. This band reacts with antibodies prepared
against the GC-domain of IF-2
(Fig. 2B, lane 1). A significant amount of a form migrating at 72 kDa
can be observed especially at longer times of induction. These
observations suggest that the precursor form of IF-2
is
subjected to proteolysis in the cell or that an internal start site in
the gene is being used.
Figure 2:
Analysis of the expression of various
forms of bovine IF-2 by SDS-PAGE. A, Cells were
grown and induced, and extracts were made as described under
``Experimental Procedures.'' Samples obtained following
Ni-NTA chromatography were analyzed by SDS-PAGE. The precursor and
mature form were analyzed on a 7.5% gel; and the NG-, G-, GC-, and
C-domains were on a 12% gel. - represents no induction; +
indicates induction. The arrows indicate the positions of the
full-length precursor and mature forms of IF-2
. B, Western blot analysis of the expression of bovine
IF-2
. Lanes 1 and 2, 10-µl samples
of induced cells carrying the precursor and mature constructs,
respectively, prepared under denaturing conditions (0.1% SDS). Lane
3, IF-2
prepared under native conditions and purified
by Ni-NTA chromatography (5 µg). C, Western analysis of
the expression of various domains of IF-2
. Lanes
1-4 correspond to NG-, G-, C-, and GC-domain of
IF-2
, respectively.
When the mature form of IF-2 is
expressed, three major bands of protein are observed (Fig. 2A). The highest molecular mass form (indicated
by a small arrow) migrates at 85 kDa, the same apparent molecular mass
as IF-2
purified from liver. Shorter forms of IF-2
migrate at 80 kDa and 72 kDa on SDS-PAGE. These bands react with
the antibody prepared against the GC-domain of IF-2
(Fig. 2B, lane 2). When the clone
encoding the mature form was expressed for 1 h, two major bands were
observed corresponding to the mature form (85 kDa) and to the 80-kDa
form observed in cell extracts prepared under denaturing conditions.
The 72-kDa form of IF-2
was present only in small amounts
after a 1-h induction. When IF-2
was purified by Ni-NTA
affinity chromatography under native conditions, most of the mature
form of this factor was degraded into the species migrating at 72 kDa
or into even shorter fragments (Fig. 2B, lane
3), suggesting that it was being degraded by proteases in the cell
extract.
The NG-, G-, GC-, and C-domains were also expressed in E. coli. Their apparent sizes on a 12% SDS-PAGE gel are 38,
25, 69, and 52 kDa, respectively (Fig. 2A). The
apparent molecular masses of these expressed proteins on SDS-PAGE
appear to be 5 kDa larger than the calculated value presumably
partially due to the His tag. A shorter form of the NG-domain (33 kDa)
was also observed (Fig. 2A). Some degradation products
were also found when the G-, GC-, and C-domain were expressed.
The
antisera prepared against the GC-domain of IF-2 reacted
with the precursor and mature forms of IF-2
(Fig. 2B), the GC-domain and the C-domain (Fig. 2C). However, the antibodies did not react with
the NG-domain or the G-domain (Fig. 2C). Therefore, the
epitopes of IF-2
appear to be concentrated in the
COOH-terminal region of this protein. This observation is not
surprising since the sequence of bovine IF-2
between
Asp-429 and Glu-512 has 60% charged residues and has the highest
surface probability in the whole molecule.
Figure 3:
Purification of various forms of bovine
IF-2. A, elution profile of bovine IF-2
on Tskgel DEAE-5PW HPLC. The mature form of bovine IF-2
initially purified by Ni-NTA affinity chromatography was
subjected to chromatography on a Tskgel DEAE-5PW column as described
under ``Experimental Procedures.'' Aliquots (20 µl) of
various fractions were tested for IF-2
activity (
).
The absorbance at 280 nm was monitored (solid line) with an
ISCO UA-5 absorbance monitor on a 0.5 scale, and the column was
developed with a salt gradient (dashed line). B,
elution profile of bovine IF-2
on Tskgel SP-5PW HPLC.
Bovine IF-2
purified by Tskgel DEAE-5PW chromatography was
subjected to chromatography on a Tskgel SP-5PW column. Aliquots (20
µl) of various fractions were tested for IF-2
activity
(
). The absorbance at 280 nm was monitored (solid line)
with an ISCO UA-5 absorbance monitor on a 0.1 scale and the column was
developed with a salt gradient (dashed line). C, the
purity of mature bovine IF-2
and the GC-domain was
analyzed by 10% SDS-PAGE. Lane 1, sample of the preparation
obtained from Ni-NTA affinity chromatography (4.4 µg). Lane
2, sample from the Tskgel DEAE-5PW preparation (1.2 µg). Lanes 3-6, sample from peaks 1-4 of bovine
IF-2
obtained from the Tskgel SP-5PW column. Lane
3, IF-2
S1 (
0.4 µg); lane 4,
IF-2
L (
0.4 µg); lane 5,
IF-2
S2 (
0.1 µg); lane 6,
IF-2
S3 (
0.2 µg). Lane 7, the purified
GC-domain of IF-2
(0.72
µg).
IF-2 was further purified by chromatography on a Tskgel
DEAE-5PW HPLC column (Fig. 3A). The IF-2
activity eluted from this column at about 0.15 M KCl.
This procedure resulted in approximately a 2-fold purification of
IF-2
activity with 53% recovery of activity (Table 1).
Finally, the sample was purified by chromatography
on a Tskgel SP-5PW column. Four peaks with IF-2 activity
were separated by this procedure (Fig. 3B). Analysis of
the material in these peaks by SDS-PAGE indicated that peaks
1, 3, and 4 all contain the 72-kDa form of
IF-2
. These forms (designated IF-2
S1, S2, and
S3, respectively) have almost identical molecular masses on SDS-PAGE.
The second peak contains the 80-kDa form of IF-2
(IF-2
L). The purity of all of these forms was
estimated to be >85% (Fig. 3C, lanes
3-6). The overall purification procedure resulted in about a
1000-fold purification with a 32% yield of the initial IF-2
activity. All of the forms of IF-2
obtained were
active in promoting the binding of fMet-tRNA to the small ribosomal
subunit of either E. coli or bovine mitochondrial ribosomes.
As indicated in Table 1, IF-2
L, IF-2
S1,
and IF-2
S2 all appear to be as active as the native bovine
IF-2
purified from liver which has a specific activity of
4900 units/mg(1) . The specific activity of IF-2
S3
seemed to be higher than that of the other purified forms. However,
since the amount of IF-2
S3 was very small, the protein
concentration in the sample was difficult to determine accurately.
To clarify the relationship between the various forms of
IF-2, purified proteins were subjected
NH
-terminal sequence analysis (Table 2).
NH
-terminal analysis of the 80-kDa form
(IF-2
L) suggested that it arose from the use of the
Met-106 codon as an internal start site (Table 2). This codon has
a potential Shine-Dalgarno sequence 12 nucleotides upstream of an AUG
codon. The distance between the Met-106 AUG codon and the putative
Shine-Dalgarno sequence is longer than usual (12 nucleotides as opposed
to an average of 7 nucleotides 5` to the start codon). However, the
mRNA may lack secondary structure in this region allowing the 30 S
subunit to bind and initiate. It has been proposed that efficient
initiation in unstructured regions of mRNAs does not require a strong
Shine-Dalgarno sequence(16) .
It is also possible that
IF-2L could arise from proteolysis since a form of E.
coli IF-2, IF-2
(65 kDa), is found to be the result of
cleavage of IF-2
by the outer membrane protease OmpT(17) .
OmpT is an endoprotease associated with the outer membrane in E.
coli K-12, which specifically hydrolyses peptide bonds between
consecutive basic residues (Lys-Lys, Lys-Arg, Arg-Lys, and
Arg-Arg)(18, 19) . However, IF-2
L was
produced in similar amounts when expressed in either E. coli M15 or in E. coli BL-21(DE-3) (data not shown). The
latter strain is deficient in the lon and OmpT proteases. These
observations suggest that IF-2
L is not the product of
proteolysis in the cell.
The major species of the shorter forms of
IF-2 (IF-2
S1) begins with the lysine at
position 154 of the full-length amino acid sequence (Table 2).
The minor species, IF-2
S2, begins with a methionine
inserted in response to the Met-147 codon while IF-2
S3
begins with a Met inserted in response to the Val-141 codon (Table 2). IF-2
S1 and IF-2
S2 most
likely result from proteolysis during purification or in the cell
itself. IF-2
S1 does not begin with Met and the sequence of
the cDNA in this region does not contain an initiation codon or a
Shine-Dalgarno sequence. The amount of this species increases
considerably during purification under native conditions. Although the
minor form IF-2
S2 begins with Met, this residue is not
proceeded by a Shine-Dalgarno sequence. This form also probably arises
from proteolysis. The minor short form, IF-2
S3, appears to
arise from the use of an internal GUG codon as an initiation codon
since Val-141 is replaced by methionine at its NH
terminus (Table 2). The size of IF-2
L (622 amino acids)
resembles the mature form of yeast IF-2
, while
IF-2
S1 (574 amino acids), IF-2
S2 (581 amino
acids), and IF-2
S3 (587 amino acids) are similar in size
to Thermus thermophilus IF-2 (572 amino acids)(4) .
The GC-domain was also purified under conditions similar to those
used for IF-2. This derivative of IF-2
was
active in promoting fMet-tRNA binding to mitochondrial and E. coli ribosomes. Two mg of the GC-domain with a purity of >95% were
obtained from about 24 g of induced E. coli cells after
purification by the Ni-NTA affinity chromatography followed by anion
and cation exchange chromatography on HPLC (Fig. 3C, lane 7).
The activities of IF-2L,
IF-2
S1, and the GC-domain have been compared in more
detail (Fig. 4). All of these proteins are active in promoting
fMet-tRNA binding to mitochondrial 28 S ribosomal subunits,
mitochondrial 55 S ribosomes and E. coli 70 S ribosomes.
IF-2
S1 has about the same activity as IF-2
L.
The GC-domain is
50% as active as IF-2
S1. These
results suggest that the 20 amino acid residues of IF-2
S1
proceeding the GC-domain are somewhat important for the function of
IF-2
although the 20 amino acid residues are not conserved
among different IF-2 s. The activities of IF-2
S1,
IF-2
L, and the GC-domain have also been determined in the
presence of either GTP or its nonhydrolyzable analog GMP-PNP. The
activities of these factors decrease 2-3- fold in the presence of
GMP-PNP, suggesting that all of these forms of IF-2
can be
recycled under the assay conditions used (data not shown).
Figure 4:
Comparison of the activities of
IF-2S1, IF-2
L and the GC-domain. The activity
of various forms of bovine IF-2
was determined as
indicated under ``Experimental Procedures.'' A,
reaction mixtures (100 µl) contained 0.25 A
units of bovine mitochondrial 28 S ribosomal subunits, the
indicated amounts of IF-2
S1 (
), IF-2
L
(
), or the GC-domain (
). B, reaction mixtures
contained 0.25 A
of mitochondrial 55 S ribosomes
and indicated amounts of IF-2
S1 (
),
IF-2
L (
), or the GC-domain (
). C,
reaction mixtures contained 30 µg of E. coli 70 S
ribosomes, 2 units of E. coli IF-3, 1 unit of E. coli IF-1, and indicated amounts of IF-2
S1 (
),
IF-2
L (
), or the GC-domain
(
).
Partially
purified NG-, G-, and C-domains were not active in promoting fMet-tRNA
binding to ribosomes indicating that both the G- and C-domains are
essential for IF-2 activity.
Figure 5:
Binding of various forms of IF-2 to mitochondrial ribosomes. Airfuge analysis of initiation
complexes was carried out as indicated under ``Experimental
Procedures.'' Lane 1, mitochondrial ribosomes alone
without IF-2
; lane 2, 58.7 pmol of
IF-2
S1; lane 3, 46 pmol of IF-2
L; lane 4, 103 pmol of the GC-domain; lane 5, 186 µg
of partially purified C-domain. Ribosome complexes were separated by
Airfuge centrifugation and tested for IF-2
by Western
analysis.
Interestingly the C-domain of
IF-2 clearly bound to 55 S ribosomes (Fig. 5, lane 5), although it did not promote fMet-tRNA binding to the
ribosomes. Gualerzi et al.(20) have proposed that a
portion of the C-domain (24 kDa) of B. stearothermophilus IF-2
corresponding to domain VI of E. coli IF-2 contains the
fMet-tRNA binding site, but has negligible binding to ribosomes. The
C-domain of IF-2
under study here contains both domains V
and VI of E. coli IF-2
. Therefore, the region
corresponding to domain V (Met-331-Lys-461) may contain the
ribosome binding site.
The roles of guanine nucleotides, fMet-tRNA,
and poly(A,U,G) in the binding of IF-2 to ribosomes was
examined again using Airfuge centrifugation. As indicated in Fig. 6A (lane 1), a significant amount of
IF-2
L could be detected bound to ribosomes in the absence
of other components of the initiation machinery. The presence of the
GTP analog, GMP-PNP, enhanced the binding IF-2
to the
ribosomes about 3-fold (Fig. 6A, lane 2). No
further enhancement of binding was observed upon the addition of
fMet-tRNA and poly(A,U,G) (compare lanes 2 and 3).
These observations suggest that IF-2
has an intrinsic
affinity for the ribosome and that this affinity is enhanced by the
presence of the GTP analog.
Figure 6:
Binding of IF-2L to
mitochondrial 55 S ribosomes. A, airfuge centrifugation was
carried out as described under ``Experimental Procedures.'' Lane 1, IF-2
L (46 pmol) and ribosomes only; lane 2, IF-2
L, ribosomes and GMP-PNP; lane
3, complete system including fMet-tRNA and poly(A,U,G). B, the effect of guanine nucleotides on the binding of
IF-2
L to mitochondrial 55 S ribosomes. Airfuge
centrifugation was carried out in the presence of 46 pmol of
IF-2
L, 1.5 A
of 55 S ribosomes, and
0.4 mM of the indicated guanine nucleotide. No fMet-tRNA and
poly(A,U,G) were added. Lane 1, no nucleotide was added; lane 2, GMP-PNP; lane 3, GTP, 4 mM phospho(enol)pyruvate, and 0.4 unit of pyruvate kinase; lane
4, GDP.
The effects of GDP, GTP, and GMP-PNP on
the binding of IF-2 to 55 S ribosomes were then
determined. Again, the binding of IF-2
could be detected
in the absence of added nucleotide (Fig. 6B, lane
1). A substantial enhancement of IF-2
binding to
ribosomes was, once again, observed with GMP-PNP (Fig. 6B, lane 2). GTP also strongly enhanced
the binding of this factor to ribosomes (lane 3).
Surprisingly, enhanced binding of IF-2
to ribosomes was
also observed in the presence of GDP although less binding was observed
in the presence of GDP than in the presence of GTP (Fig. 6B, lane 4). GTP hydrolysis is thought
to facilitate the release of IF-2 from the ribosome(21) . The
formation of the IF-2
GDP complex on the ribosome
following GTP hydrolysis may weaken the interaction of IF-2
with the ribosome sufficiently to result in the dissociation of
the IF-2
GDP complex. Alternatively, the dissociation
of GDP from the IF-2
GDP complex while still on the
ribosome could result in a conformational change on IF-2
that further weakens its interaction with the ribosome and
IF-2
would then dissociate.
It has been reported that E. coli IF-2 binds to ribosomal particles with decreasing affinity: 30 > 70 > 50 S(22) . GTP and GDP have no effect on the binding of E. coli IF-2 to 70 S ribosomes(23) . GTP stimulates the binding of this factor to the 30 S subunit and somewhat decreases its binding to 50 S subunits; GDP has the opposite effect. These results, and the data presented here, suggest that the dissociation of GDP from IF-2, while the factor is still on the ribosome, results in a conformation with a lower affinity for the ribosome, thus, promoting the release of the factor.
The formation of
an IF-2fMet-tRNA complex was also weak. The K
for the formation of a binary complex
(IF-2
fMet-tRNA) was estimated to be about
10
-10
M
(data
not shown). The formation of this complex was inhibited by
Mg
suggesting that it does not play a physiological
role in initiation. GTP did not affect the formation of the
IF-2
fMet-tRNA complex. Hence, a ternary
IF-2
fMet-tRNA
GTP complex does not appear to
play a role in initiation in the animal mitochondrial system. These
data and the results of the Airfuge centrifugation studies described
above suggest that IF-2
binds to mitochondrial ribosomes
prior to its interaction with the initiator tRNA or GTP.