(Received for publication, July 25, 1994; and in revised form, November 18, 1994)
From the
Complete cDNAs encoding human mitochondrial translational
initiation factor 2 (IF-2) have been obtained from liver,
heart, and fetal brain cDNA libraries. These cDNAs have a long open
reading frame 2181 residues in length encoding a protein of 727 amino
acids. Overall, human IF-2
has 30-40% identity to
the corresponding prokaryotic factors. Surprisingly, it is no more
homologous to yeast IF-2
than to the IF-2s from bacterial
sources. The greatest region of conservation lies in the G-domain of
this factor with less conservation in the COOH-terminal half of the
protein and very little homology near the amino terminus. The
5`-untranslated leaders of the liver and heart cDNAs contain a number
of short open reading frames. These sequences may play a role in the
translational activity of the IF-2
mRNA. Northern analysis
indicates that the IF-2
gene is expressed in all tissues
but that the level of expression varies over a wide range.
The initiation of protein biosynthesis has been extensively
studied in prokaryotes and in the cytoplasm of eukaryotic
cells(1, 2, 3, 4, 5) .
During the initiation process, initiation factor 2 promotes the binding
of the initiator tRNA to the small subunit of the ribosome in a
GTP-dependent manner. Prokaryotic initiation factor 2 (IF-2) ()is a single polypeptide chain having a molecular weight of
78,000-97,000. It promotes fMet-tRNA binding to 30 S ribosomal
subunits in a mRNA-dependent reaction. Eukaryotic cytoplasmic
initiation factor 2 (eIF-2) is a trimeric protein with a molecular
weight of 145,000(3) . It is responsible for the binding of
Met-tRNA to 40 S subunits. This reaction is believed to occur prior to
the interaction of the subunit with mRNA(3) .
The mechanism
of translational initiation in animal mitochondria is not yet
understood, but several studies suggest that it will have a number of
unique features(6, 7, 8, 9) . To
date, only one initiation factor has been identified in the animal
mitochondrial system(10, 11) . This factor, which was
purified from bovine liver, is equivalent to prokaryotic IF-2 and has
been designated IF-2(11) . It is a monomeric
protein with a molecular weight of 85,000. IF-2
promotes
the binding of fMet-tRNA to animal mitochondrial ribosomes in a GTP-
and mRNA-dependent reaction.
Genes encoding IF-2 have been cloned
from several prokaryotic
microorganisms(12, 13, 14, 15) . The
cDNA sequences encoding the ,
, and
subunits of eIF-2
have also been recently
reported(16, 17, 18) . Although IF-2
in yeast has not been purified or characterized, its gene has
been cloned(19) . The sequence of this factor suggests that it
has significant homology to bacterial IF-2 except near the amino
terminus. In the present study, we have cloned and characterized the
cDNAs for IF-2
from human liver, heart, and fetal brain.
For the production of
cDNA clones, 2 µg of poly(A) RNA from human liver
was reverse transcribed with Moloney murine leukemia virus reverse
transcriptase basically as described(20) . 1 µl of the
reaction mixture was used for the initial PCR, which was carried out
using primers P1F and P1R. A portion (2 µl) of the first PCR
reaction mixture was then amplified using the nested primers P2F and
P2R. PCR reactions were analyzed on 4% NuSieve-agarose gels, and the
products were purified from the gel using silicon powder
(Sigma)(21) . DNA fragments were digested with BamHI
and EcoRI and cloned into pTZ19R (Pharmacia Biotech Inc.) (22) The plasmid containing the cloned portion of the
IF-2
cDNA is designated pTZIF2G, and the insert contained
in this plasmid is designated IF2G when referred to below.
Figure 1:
Conserved sequences in IF-2 and cloning
strategy. The primer P2F was designed based on a sequence conserved in
prokaryotic IF-2s. Primer P1R was based on a sequence found in yeast
IF-2 and in a peptide obtained from bovine
IF-2
. Primers P1F and P2R were based on sequence
information obtained with bovine IF-2
. Primer P1R was used
to prepare a specific cDNA using human liver poly(A)
RNA and reverse transcriptase. Two rounds of PCR were then
carried out using the indicated primers.
To ensure that this band was indeed derived from
the IF-2 cDNA, a second round of PCR was carried out. This
step was based on the observed sequence conservation of the IF-2s from
various organisms (Fig. 1). The peptide sequences of the factors
from Escherichia coli(12) , Bacillus
stearothermophilus(14) , Streptococcus
faecium(15) , and Bacillus subtilis(13) , along with Saccharomyces cerevisiae IF-2
(19) , were analyzed using the PILEUP
program in the GCG software package. The sense primer P2F was designed
from one of these sequences, PVVTIMG, which marks the amino-terminal
boundary of the G-domain of prokaryotic IF-2 (Fig. 1). A second
round of PCR was carried out using a nested set of primers lying inside
the first pair of primers (the P2F primer derived from sequences
conserved in IF-2s and P2R derived from peptide sequence information
obtained from bovine IF-2
). Gel analysis indicated that
the major product corresponded well to the size predicted to span the
region between the selected primers. The product was cloned and
sequenced. The amino acid sequence deduced from the cDNA clone shares
59% identity to the yeast IF-2
and 69% identity to E.
coli IF-2. It exhibits over 90% identity to the bovine IF-2
peptide sequences shown in Fig. 1. It has 27% sequence
identity to eIF-2
but no significant homology to either the
or
subunits of eIF-2. The cDNA obtained, therefore, most likely
encodes a portion of the G-domain of human liver IF-2
.
The liver IF-2 cDNA was used to screen a human liver
oligo(dT)-primed
Uni-ZAP cDNA library. 16 positive clones with
inserts ranging in size from 500 to 2500 bp were purified, and the
plasmids containing the inserts were excised in vivo. The
largest clone characterized carries a 2541-bp-long insert and was
sequenced in both directions. This clone is derived from a mRNA
containing a 270-base pair 5`-untranslated leader (UTL), a 2181-base
pair open reading frame encoding IF-2
, a 26-base pair
3`-untranslated leader, and a poly(A) tail of over 60 residues (Fig. 2). Recently, cloning of human infant brain-expressed
sequence tags has yielded two clones containing sequences encompassing
about 165 amino acids that are homologous to bacterial
IF-2s(25) . These expressed sequence tag clones are
98-99% identical to regions in the G-domain and near the COOH
terminus of the human IF-2
cDNA identified here.
Therefore, we believe that these expressed sequenced tags are derived
from human IF-2
cDNAs.
Figure 2:
Nucleotide and deduced amino acid
sequences of human liver IF-2. Sequences in the
5`-untranslated leader are designated as negativenumbers. The nucleotide numbered +1 corresponds to
the first base of the initiation codon ATG at the beginning of the
longest open reading frame. The putative polyadenylation signals are underlined, and (A)
denotes the poly(A) tail. The
proposed processing site for the mitochondrial processing endopeptidase
is indicated by a solidinvertedtriangle.
Human heart and human fetal
brain cDNA libraries were also screened, and IF-2 cDNA
clones were isolated from them. The complete nucleotide sequences of
two clones with 2.5-kb inserts were obtained from the heart and fetal
brain libraries. The IF-2
cDNAs from heart and fetal brain
share essentially the same coding sequence and 3`-untranslated regions.
The heart and liver cDNA share the same 5`-untranslated leader except
that the heart IF-2
cDNA has a slightly different sequence
at the very 5`-end. The 5`-UTL of the fetal brain IF-2
cDNA, however, is very different from those found in the heart
and liver clones. It is possible that this difference may have arisen
from the transcription of separate genes or may be the result of the
cloning process.
The long open reading frame is 2181
residues long and encodes a 727-amino acid protein. The translational
initiation codon for IF-2 has been tentatively identified
as the first in-frame AUG of this long open reading frame. The
3`-untranslated region of the cDNA is unusually short, and this
26-bp-long sequence is 85% A/T. As shown in Fig. 2, two
sequences, ATTAAA and AATAAC, upstream from the poly(A) tail resemble
the consensus sequence AATAAA for polyadenylation(30) .
Therefore, one or both of them is probably used as a polyadenylation
signal for the human IF-2
gene. It has been reported that
the eIF-2
gene may also use ATTAAA as the signal for
polyadenylation(17) .
Most mitochondrial proteins are
synthesized as precursors on cytosolic polysomes and are subsequently
imported into mitochondria. The vast majority of these precursors carry
amino-terminal presequences, which contain the information required for
their targeting to mitochondria. However, no significant sequence
homology has been found in the mitochondrial targeting sequences. In
general, presequences of mitochondrial proteins are rich in positively
charged residues, lack acidic residues, and most have a high content of
hydroxylated residues (31) . In addition, many of these
sequences appear to be able to form amphiphilic helices. The
first 30 amino acids of human liver IF-2
from the amino
terminus contain a number of basic residues, including 2 lysines, 4
arginines, and 2 hydroxylated amino acids. The mitochondrial targeting
sequence at the amino terminus of human IF-2
is predicted
to form an amphiphilic
helix between residues 1 and 15. A
putative cleavage site is predicted between Ala-29 and Leu-30 based on
the analysis of cleavage patterns in other mitochondrial import
sequences.
The predicted mature form of IF-2 consists
of 698 amino acid residues and has a molecular weight of 77,500. Its
isoelectric point is calculated to be 6.6. Previous work has indicated
that bovine IF-2
has an apparent molecular weight of
85,000 on SDS-polyacrylamide gel electrophoresis. Thus, human and
bovine IF-2
appear to be similar in size. Human liver
IF-2
is similar to prokaryotic IF-2 (except E. coli IF-2
) in size. It is about 50 residues longer than yeast
IF-2
, assuming that the transit sequences are about the
same length(19) .
As indicated in Fig. 3and Table 1, the peptide sequence of the mature form of human
IF-2 displays extensive homology to the COOH-terminal two
thirds of prokaryotic IF-2s. However, it shows no significant
similarity to the amino-terminal peptide sequences of these factors.
The amino-terminal peptide sequence (Leu-30 to Ser-180) of the mature
human IF-2
has 29% charged residues. The corresponding
regions in the IF-2 of E. coli or yeast mitochondria contain
approximately 26% charged residues. Using Chou-Fasman and
GarnierOsguthorpe-Robson methods, we predict that both the human
mitochondrial factor and E. coli IF-2 have similar secondary
structures at this region(32, 33) .
Figure 3: Comparison of primary sequences of IF-2 from B. stearothermophilus (Bstea), B. subtilis (Bsubt), S. faecium (Sfaec), E. coli (Ecoli), human liver mitochondria (Mthum), and S. cerevisiae mitochondria (Mtyst). The sequence alignment was made using PILEUP from the GCG computer software package, and the conserved residues shaded in black were made using BOXSHADE (netserv@EMBL-Heidelberg.de).
There is
striking primary sequence homology in the Gdomain of IF-2, the central
domain corresponding to residues 392-540 of E. coli IF-2 and residues 181-330 of IF-2
. The
central domain of human IF-2
exhibits an average 67%
identity to prokaryotic IF-2s (Table 1). However, it has only 59%
identity, the lowest among those compared, to its yeast mitochondrial
counterpart. The alternative
sheet/
helix secondary
structure predicted to be present in the G-domain of IF-2 is retained
in the central domain of human IF-2
(33) . The
similarity between human IF-2
and other IF-2s decreases
remarkably at the COOH terminus, the region thought to be the tRNA
binding domain. As shown in Table 1, human IF-2
has
an average of 35% identical residues with other IF-2s within this
region. In contrast to other IF-2s, the region from Asp-429 to Glu-512
in the COOH terminus of human IF-2
has the highest surface
probability in the whole molecule. Over 50% of the amino acid residues
within this region are charged. It also has an extra 37 amino acid
residues not found in any prokaryotic IF-2. These additional amino
acids (Fig. 3) are clustered primarily in three closely spaced
sections near the beginning of the COOH-terminal half of the protein.
Two of these regions are also observed in yeast IF-2
. In
general, human IF-2
shows an overall 36% sequence identity
to IF-2 from prokaryotes and yeast mitochondria. The observations
summarized above suggest that the G-domain of IF-2 has been highly
conserved during evolution while other regions of the molecule have
been allowed to diverge to a greater extent.
Figure 4:
Northern analysis of the expression of
IF-2 mRNA in different human
tissues.
In addition to the
major IF-2 mRNA observed at 2.5 kb, two other bands of
approximately 3.5 and 1.5 kb, respectively, were observed on the
Northern blot (Fig. 4). These bands are present at about 5% of
the intensity of the prominent 2.5-kb transcript and may represent
signals from a related gene. Alternatively, they could arise from a
precursor and a degradation product, respectively, of the IF-2
mRNA.
Figure 5:
Southern analysis of the presence of
sequences related to IF-2 in different
species.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L34600[GenBank].