Genomic Cloning and Characterization of the Human Eukaryotic
Initiation Factor-2
Promoter*
John A.
Chiorini,
Suzanne
Miyamoto,
Stephanie J.
Harkin, and
Brian
Safer
From the Molecular Hematology Branch, NHLBI, National Institutes of
Health, Bethesda, Maryland 20892
 |
ABSTRACT |
The translation initiation factor eIF2 consists
of three subunits that are present in equal molar amounts. The genomic
DNA containing the gene for eIF2
and its promoter were cloned and sequenced to characterize further the mechanism of their regulated synthesis. Whereas Southern blot analysis indicated that a number of
copies of the gene may exist, only one full-length intron-containing copy was identified. Similar to the eIF2
promoter, the eIF2
promoter is TATA-less, CAAT-less, and GC-rich and contains
an
-Pal binding motif. Mutation of the
-Pal binding sequence
resulted in an 8-fold decrease in activity when assayed by the
luciferase reporter gene constructs. The data suggest a common
mechanism of transcriptional control for the two cloned subunits of eIF2.
 |
INTRODUCTION |
The translation initiation factor eIF2 catalyzes the first
regulated step of protein biosynthesis, the binding of the initiator Met-tRNAi to the 40 S ribosomal subunit. Binding occurs as
a ternary complex of Met-tRNAi-eIF2-GTP. All three subunits
of eIF2 (
,
,
) are required for the catalytic utilization of
eIF2 during protein synthesis initiation (1, 2). The
subunit of
eIF2 is a 36.2-kDa polypeptide whose phosphorylation state regulates activity of the heterotrimer (3). The
subunit appears to bind GTP
or GDP and is a 51.9-kDa polypeptide (4). The 38.3-kDa
subunit may
be directly involved in binding of the ternary complex to mRNA (5,
6). None of the three subunits appears to exist as a monomer outside
the eIF2 heterotrimer. Initial experiments demonstrated that balanced
synthesis of the
and
subunits is predominantly the result of
different rates of ribosomal elongation (7). Subsequent experiments
suggested that eIF2
expression could be regulated by antisense
transcripts that form double-stranded RNA during T-cell activation (8).
Regulation of sense transcription is under the control of a
transcription factor designated
-Pal, which binds to an unusual
direct repeat element.
-Pal is homologous to the developmental
transcription factors P3A2 and ewg (9). Potential target
genes of
-Pal can be broadly classified as encoding growth-responsive factors (9). To see if the eIF2
subunit was
regulated via similar mechanisms, the eIF2
gene and its promoter region were cloned and characterized.
 |
MATERIALS AND METHODS |
Library Cloning Screening--
The cDNA for eIF2
was a
gift from J. W. B. Hershey (GenBank number M29536). The nucleotide
sequence of the promoter region has been reported to GenBank (number
AF076927). A human lung fibroblast genomic library was constructed in
the
phage vector. Lambda Fix was purchased from Stratagene (La
Jolla, CA). Subgenomic libraries were constructed by digesting high
molecular weight DNA from K562 cells (ATCC, Manassas, VA) to completion
with the indicated restriction enzymes followed by separation on a
0.5% Sea Kem GTG-agarose gel (FMC, Rockland, ME) and staining with ethidium bromide, and was the region containing the fragment of interest excised. The DNA was then purified and ligated into prepared phage arms following the manufacturer's instructions (Stratagene). To
ensure the proper region of the gel was excised, the gel (minus the
excised band) was Southern blotted and probed with the fragment of
interest. Screening of these libraries was carried out essentially as
described previously (10). Northern blots were prepared following standard procedures (10). Southern blots of genomic DNA were prepared
following the instructions for use of the Zeta probe blotting membranes
(Bio-Rad). All hybridizations were done in Church-Gilbert buffer (0.5 M NaPO4 (pH 7.0), 1% bovine serum albumin, 7%
sodium dodecyl sulfate, 10 mM EDTA) for 18 h at the
indicated hybridization temperature and washed with a final stringency
of 0.15 M NaCl for 1 h at the indicated hybridization temperature.
Primer Extension Analysis--
Primer extension analysis
was performed using MLV reverse transcriptase as described by the
manufacturer (Life Technologies, Inc.). To determine the size of the
products, four sequencing reactions were performed and run on the same gel.
Ribonuclease Protection Assays--
Ribonuclease protection
assays (RPAs)1 were performed
as described by the manufacturer (Ambion, Austin, TX). Riboprobes were generated using the Maxiscript in vitro transcription kit
according to the manufacturer's instruction (Ambion). The size of the
protected fragments was determined by comparison with control RNA and
by comparison with a dideoxy sequencing ladder.
Luciferase Assay--
Luciferase assays were performed as
described previously (11). Briefly, NIH 3T3 cells were plated in 6-well
plates at a density of 1 × 105 cells/well and
transfected using LipofectAMINE (Life Technologies, Inc.). After
48 h, cell lysates were prepared, and 5-µl aliquots were added
to 100 µl of luciferase reagent and assayed in a MonolightTM 2010 luminometer for 10 s. Final results were given in units of luciferase activity/µg of protein.
 |
RESULTS |
A cDNA clone for eIF2
was isolated from human liver
mRNA, and the encoded polypeptide was shown to interact with the
eIF2
and
subunits (6). Southern blots of DNA isolated from K562 cells (ATCC) probed with the eIF2
cDNA produced a large number of fragments, indicating the gene for eIF2
is either very large or
there are multiple copies (Fig.
1A). Hybridization of
duplicate Southern blots with either an oligonucleotide probe from the
3'-UTR (bases 1273-1298) or a random-primed fragment from the 5'-UTR (1-103) generated a simplified pattern of only 4 or 5 bands (Fig. 1,
B and C, respectively). These results suggest the
existence of multiple genomic fragments with a high degree of homology
to the eIF2
cDNA. Because of the high degree of specificity of
oligonucleotide probes compared with random-primed fragments, the
3'-UTR oligonucleotide probe was initially used to screen a human
fibroblast genomic library.

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Fig. 1.
eIF2 Southern
analysis. A, hybridization of K562 genomic DNA with
random-primed cDNA. M, markers; 1, 10 µg of
DNA digested with EcoRI; 2, 10 µg digested with
HindIII. B, hybridization of K562 genomic DNA
with an oligonucleotide probe from the 3'-UTR (1273-1298). 10 µg of
DNA was digested with the following enzymes: 1,
EcoRI; 2, HindIII; 3, PstI; 4, XbaI; 5, EcoRI. C, hybridization of K562 genomic DNA
digested with EcoRI and probed with a random-primed probe
from the 5'-UTR (1-103).
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Genomic clones corresponding to three of the four bands detected using
the 3'-UTR oligonucleotide probe were isolated after screening multiple
libraries (Fig. 2). Although all three
exhibited a high degree of homology to the cDNA, only one was
nearly identical and contained introns. This clone is referred to as
clone 1A (Fig. 2). Additional sequencing and restriction mapping showed
that clone 1A contained an insert 13 kilobases in length and 550 bases of the cDNA divided into 5 exons. A full-length clone was obtained by using polymerase chain reaction to amplify the DNA between the last
exon in clone 1A and upstream exons. The promoter region and initiation
codon were cloned by creating subgenomic libraries of the five DNA
fragments that hybridized to the 5' 103 base pairs of the cDNA as
shown in Fig. 1C. From the subgenomic library constructed from the 2.3-kilobase fragments, one clone was isolated that contained the missing cDNA sequence and could be linked to clone 1A. This produced a full-length cDNA and is referred to as clone 1 (Fig. 2).
In addition, another pseudogene was isolated from this library, which
contained the whole open reading frame with a number of point mutations
but lacked introns. This clone is referred to as clone 4 (Fig.
2).

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Fig. 2.
cDNA maps. Sequenced regions of the
eIF2 genes are depicted below the published cDNA. The
arrows indicate the beginning and the end of the coding
region of the cDNA. The dots above the lines
indicate the regions of sequence divergence from the published cDNA
sequence.
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The sequence of clone 1 differed from the published cDNA in two
respects. The 5' most 18 bases reported to be in the cDNA were not
present in the 2.3-kilobase promoter fragment. Clone 1 also differed
from the cDNA at position 1000. The cDNA contained a T at this
position and the genomic clone contained a C. This difference could be
the result of a mutation introduced by reverse transcriptase during the
cloning of the eIF2
cDNA or it could indicate that more than one
copy of the
gene exists. To distinguish between these two
possibilities, three identical Northern blots were prepared and probed
with complementary oligonucleotide probes that contained the genomic
version of this sequence based on clone 1 (5'-AGAGTCTGTCCTTC), the
cDNA version of the sequence (5'-AGAGTCGTATGTCCTTC), or a unique
sequence found in one of the pseudogenes, clone 2 (5'-TCACATGAAACTATTAAGTAAGC). Under stringent washing conditions each
oligonucleotide probe hybridized to the plasmid from which its sequence
was obtained (Fig. 3). Although the
cDNA and clone 1 oligonucleotide probes differed by a single
nucleotide, no cross-hybridization was observed in the other plasmid.
When these same hybridization conditions were used to probe Northern
blots, only the oligonucleotide from clone 1 hybridized to a message on
the Northern blots (Fig. 4B).
The mRNAs detected by the clone 1 oligonucleotide probe are of
similar mobility to those detected by hybridization with the full-length cDNA (Fig. 4D). Therefore clone 1A would
appear to contain the correct sequence. Because the oligonucleotide
probe based on the published cDNA sequence did not hybridize to the Northern blot (Fig. 4A), the T at position 1000 may be an
artifact introduced during the cloning process of the cDNA.

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Fig. 3.
Oligonucleotide hybridization
conditions. Complementary oligonucleotide probes corresponding to
the divergent region in the coding region of the cDNA (991-1008)
and the corresponding sequence in clone 1 and clone 2 were synthesized
and used to probe three panels of DNA dot blots. Each panel contained a
dot of 100 ng or 10 ng of DNA of the cDNA clone 1 or clone 2 as
indicated to the left of the figure. The oligonucleotide
probe used to probe the panel is indicated at the top of the
figure. Under stringent washing conditions, the oligonucleotide probes
would only hybridize to their corresponding target DNA.
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Fig. 4.
Northern blot analysis. Similar
hybridization and washing conditions to those used in Fig. 3 were used
to hybridize and wash three Northern blots. The oligonucleotide probe
used as a probe on the blot is indicated above the blot. A,
cDNA oligonucleotide probe; B, clone 1 oligonucleotide
probe; C, clone 2 oligonucleotide probe. Within each blot
lane 1 contains 10 µg of K562 total RNA; lane
2, 0.250 µg of poly(A+) K562 RNA; lane 3,
10 µg of mouse total RNA (CLONTECH); lane
4, 0.250 µg of mouse poly(A+) RNA
(CLONTECH). A eIF2 message was only detected
with the probe derived from clone 1. No signal above background was
detected with the other two probes. D, as a size control an
additional Northern blot was probed with a random-primed probe of the
entire cDNA. Lanes 1-3 contain 5, 10, and 20 µg of
K562 total RNA, respectively.
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To define the sequence at the 5' end of the message, total K562 RNA was
hybridized with radiolabeled antisense riboprobes derived from either
the 5' end of the cDNA or clone 1 and analyzed in a RPA. As a size
control, the antisense riboprobes were also hybridized with a
non-radiolabeled sense fragment transcribed from the original eIF2
cDNA. Fig. 5A (lane
3) shows that incubation of the radiolabeled cDNA probe with
the cDNA sense control gave a band that was the correct length for
a fully protected cDNA fragment. As a point of reference the length
of this band is defined as 1 (Fig. 5, A and B).
Incubation of the cDNA sense control message with the clone 1 riboprobe produced a band 18 bases shorter that corresponds to the
point of divergence for the cDNA sequence and the genomic sequence.
The length of this band is correspondingly
18 (Fig. 5, A,
lane 11, and B). Incubation of the cDNA probe with total K562 RNA also produced bands of approximately 18 bases shorter than the cDNA/cDNA hybrid, indicating that the
5'-terminal 18 bases contained in the cDNA are not present in the
pool of K562 RNA (Fig. 5A, lanes 4-6).
Hybridization of the clone 1 riboprobe with total K562 RNA generated
bands approximately 21 bases longer than the cDNA, indicating that
the genomic sequence of clone 1 was again correct and that the 5'-UTR
was longer than previously reported (Fig. 5A, lanes
13-15). Because the cDNA was isolated from a library derived
from liver mRNA (6), this difference could be tissue-specific.
However, hybridization with liver RNA with each probe generated bands
of similar size to the K562 RNA (Fig. 5A, lanes
7-9, 16-18).

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Fig. 5.
A ribonuclease protection assay. Total
RNA from either K562 cells or mouse liver was hybridized with antisense
riboprobes from the 5'-UTR of either the cDNA or clone 1 and
digested with RNase at a 1:50 (lanes 2-4, 7,
11-13, 16) and 1:25 dilution (lanes 5, 6, 8, 9, 14, 15, 17, 18) with either 50 µg of total RNA
(lanes 4, 5, 7, 8, 13, 14, 16, 17) or 100 µg of total RNA
(lanes 6, 9, 15, 18). As controls the two riboprobes were
each hybridized with yeast tRNA (lanes 2, 12) or in
vitro transcribed sense RNA from the cDNA (lanes 3, 11). The undigested probe was loaded in lanes 1 and
10. The numbers to the left of the
figure correspond to the relative map position based on the
hybridization of the cDNA riboprobe to the in vitro
transcribed sense cDNA RNA (lane 3). The other map
positions were determined by comparison with a sequencing ladder.
B, schematic of RPA. The relative lengths of the bands
generated by the RPA are compared and indicated above the figure. The
length of the band produced by hybridizing the cDNA riboprobe with
in vitro transcript RNA from the cDNA clone is set at 1. The columns to the right of the figure indicate which probe
was used and the corresponding RNA target to which it was
hybridized.
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To more accurately map the start site of the mRNA, total RNA from
K562 cells was hybridized with a radiolabeled antisense primer binding
87 bases from the 5' end of the cDNA. The primer was extended with
reverse transcriptase, and a series of bands was detected by
autoradiography (Fig. 6, lane
1). This pattern was simplified by the addition of actinomycin D
(Fig. 6, lane 2), and a single prominent band of 108 bases
was produced, indicating the 5' end of the message was 21 bases
upstream from the 5' end of the cDNA clone. This length is in
agreement with the potential start site identified by the RPA
analysis.

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Fig. 6.
Primer extension analysis of
eIF2 mRNA in K562 cells. Total RNA
from K562 cells was isolated and annealed with a 5' end-labeled
non-coding strand primer that hybridizes to bases 70-87 of the
cDNA. This primer was extended with reverse transcriptase (M-MLV);
the resulting product was isolated and resolved on a denaturing
polyacrylamide gel as described under "Materials and Methods." The
size of the fragment was determined by comparing the resulting bands to
a sequencing ladder (lanes labeled G,
A, T, C). The extension reaction was
carried out in the absence (lane 1) or the presence
(lane 2) of actinomycin D (50 µg/ml).
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Therefore, of the four possible eIF2
genes that were identified by
Southern blotting only one contained the entire cDNA and introns.
The organization of this gene is shown in Fig.
7A. The gene for eIF2
spans
28 kilobases and is divided into 9 exons ranging in size from 50 to 480 bases. The message appears to be transcribed from a single start site
138 bases upstream of the initiation codon and contains a 3'-UTR of 280 bases. The number of nucleotides contained in each exon is summarized
in Fig. 7B, columns 1 and 2.

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Fig. 7.
Genomic map. A, the exon
portions of the eIF2 gene are depicted as boxes.
Restriction endonuclease recognition sites are indicated: R,
EcoRI; H, HindIII; X,
XbaI; P, PstI. The numbers
below correspond to the exon number. B, exon structure and
minigene divergences. Column 2 of the table lists the
nucleotides of the cDNA contained in each exon (column
1). The numbering is based on the published cDNA (6).
Column 3 lists the amino acid changes in the minigene (clone
4) compared with the corrected cDNA (clone 1).
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Further sequencing of clone 4 showed it contains several point
mutations and an in frame deletion of 18 bases (amino acids 138-143)
(Fig. 7B, column 3). In vitro
transcription and translation of clone 4 generated a protein product
very similar in size to that translated from the cDNA (data not
shown). Several attempts were made to identify a transcript for this
gene in vivo using Northern blots, RPA of K562, and
activated lymphocyte RNA, but none could be detected (data not shown).
The promoter region of eIF2
(
1000 to +1321) was searched for
transcriptional elements and was found to contain a number of potential
transcription factor binding sites (Fig.
8). The eIF2
promoter contains a
consensus sequence for the
-Pal transcription factor at
25. This
element is also present in the promoter of the
subunit of eIF2 (9).
Positioned over the cap site is a potential Sp1 site with potential
E2F, C/EBP, c-myc, and two SIF sites located downstream at
+15, +22, +253, +93, and +106, respectively. Upstream of the cap site,
potential p53, Ap2, myoD, F-Act1, and CAAT box motifs exist.

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Fig. 8.
Promoter sequence. The sequence of the
eIF2 promoter region ( 1000 to +200) is presented. An
arrow indicates the start site of transcription as mapped by
both primer extension and ribonuclease protection experiments, and the
initiation codon is italic and boxed. The -Pal
transcription factor binding site is underlined. Other
potential transcription factor binding sites are identified by
boxes. The 5'-UTR sequence identified in clone 1, which was
missing from the published cDNA, is indicated in
bold.
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Previous research has demonstrated the importance of the
-Pal site
in the regulation of the eIF2 promoter (1). To assess the importance of
the
-Pal element in the expression of the eIF2
gene, luciferase
reporter constructs were made that contained mutations in the
-Pal
region of the promoter. The
-Pal sequence was mutated from
TGCGCAGGCGCA
to
TGCAAATTGGAT, which had previously been shown not to bind the
-Pal transcription factor (7). This mutation decreased promoter function in the reporter
constructs 8-fold compared with the wild type promoter (Fig.
9, A and B,
column 2 versus 3). A similar change in activity was detected in transfection of both 3T3 and 293 cells. To detect possible antisense activity, the promoter region was cloned into the
luciferase plasmid in the antisense orientation and transfected into
293 cells or 3T3 cells. No luciferase activity was detected using a
region corresponding to the first intron (+125 to +1300) alone in the
sense or antisense orientation (Fig. 9, columns 4 and
5, respectively). Furthermore, no luciferase activity was detected using the entire promoter region (
1000 to +1300) in the
antisense orientation (Fig. 9, column 6). Experiments using reverse transcriptase-polymerase chain reaction of RNA isolated from
Go and activated T-cells also failed to detect any
antisense RNA transcription within the first intron region of eIF2,
which has been described for eIF2 (8) (data not shown).

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Fig. 9.
Luciferase reporter analysis. Several
regions of the eIF2 gene were tested for transcriptional activity
using a luciferase reporter gene assay in either 3T3 or 293 cells
(A and B). Transcriptional activity was measured
in both the sense and antisense direction. Column 1,
luciferase gene lacking a promoter (basic); column
2, eIF2 promoter bases 1000 to +125 (eIF-2beta);
column 3, eIF2 promoter with a mutated -Pal binding site
(mut aPal); column 4, eIF2 promoter sequence
+125 to +1300 in the sense orientation (intron sense);
column 5, eIF2 promoter +125 to +1300 in the antisense
orientation (intron antisense); column 6, eIF2
promoter 1000 to +1300 in the antisense orientation (eIF-2beta
antisense). RLU, relative light units.
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 |
DISCUSSION |
Cell growth and differentiation require the regulated expression
of a large number of proteins. One mechanism for achieving coordinate
regulation is through the use of a common regulatory transcription
factor. Examples of this theme are the CREB family of responsive genes
and the NF-
B family. The presence of a functional
-Pal site in
the two subunits of eIF2 that have been cloned indicates that
-Pal
may also act as a coordinating factor.
Obtaining the genomic clone for eIF2
was much more complicated than
for eIF2
. Multiple pseudogenes were identified for the eIF2
subunit as well as an intronless minigene. In contrast, eIF2
was
encoded by a single gene (12). Furthermore, several differences were
identified in the genomic clone compared with the cDNA. Through the
use of high stringency Northern blots and RPAs, the genomic clone was
shown to be the correct sequence, and no mRNA corresponding to the
cDNA sequences could be detected. The differences in the 5'-UTR and
those at base 1000 in the published cDNA compared with the genomic
sequence are most likely the result of ligation artifacts produced
during the creation of the libraries and poor fidelity of reverse
transcriptase, respectively.
The minigene identified during the cloning may be the result of a
retrotransposition event. Although in vitro translation of
this open reading frame generated a protein that was very similar in
size to that generated from the cDNA, no transcript for this gene
could be detected in vivo by Northern blot analysis, RPA in
K562, or T-cells. Other bands are present on Northern blots probed with
the eIF2
cDNA, and these mRNAs are most likely the result of
multiple polyadenylation sites as has been reported for other
translation factors (12). Although it would appear that only one
functional gene for eIF2
exists, multiple functional copies of other
translation factors have been identified (13). Recently two genomic
clones were isolated for eIF4E; one contained introns and the other was
intronless (14).
Whereas eIF2
and eIF2
are regulated at the transcriptional level
by
-Pal, the cis elements in the two promoters are distinct. The
eIF2
subunit promoter has two adjacent sites, which bind
-Pal
with different affinities. Mutation of the high affinity site decreases
expression 12-fold, whereas mutation of the lower affinity site
inhibits expression 3-fold.2
In contrast, eIF2
has only a single site whose deletion results in
an 8-fold reduction in transcriptional activity. However, both genes
are TATA-less and have the
-Pal sites positioned in the traditional
30 TATA box site. Although potential
-Pal binding sites have been
identified in a number of other genes (9), their importance has yet to
be demonstrated. Characterization of
-Pal binding sites identified
in these other growth-responsive genes is essential to defining the
role of
-Pal as a coordinating transcription factor involved in the
regulation of expression of growth response genes.
 |
ACKNOWLEDGEMENTS |
We thank J. W. B. Hershey (University of
California, Davis) for providing the original cDNA for the eIF2
clone. We also thank the members of the Molecular Hematology Branch for
critical evaluation of this paper, including H. Nagy for assistance in
the preparation of this manuscript.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Bldg. 10, Rm. 7D18,
DIR, NHLBI, Molecular Hematology Branch, MSC 1654, Bethesda, MD
20892-1654. Tel.: 301-496-1284; Fax: 301-496-9985; E-mail: besafer{at}helix.nih.gov.
The abbreviations used are:
RPA, ribonuclease
protection assay; UTR, untranslated region.
2
J. A. Chiorini, S. Miyamoto, S. J. Harkin,
and B. Safer, unpublished data.
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