Protein synthesis is promoted by a number of proteins called eukaryotic initiation factors (eIFs) ()that transiently interact with ribosomes, methionyl-tRNA, and mRNA during the initiation phase (reviewed in (1) and (2) ). The eukaryotic initiation factors have been purified mainly from mammalian cells, and their functions have been studied in vitro in reconstituted assays for initiation of protein synthesis. To better characterize the structure/function of these proteins, the cDNAs of many of the factors have been cloned and sequenced. Recently, we reported the primary structure of eIF-1A (formerly called eIF-4C) from rabbit, human, and wheat germ obtained both by amino acid sequencing of peptides and by deduction from cloned cDNAs(3) .

Human eIF-1A is a small protein with a mass of 16.4 kDa and appears not to be post-translationally modified(4) . The factor is essential for maximal protein synthesis in either mammalian or plant in vitro systems. The most pronounced effects of this protein have been observed in 80 S ribosome dissociation, stabilization of initiator Met-tRNA binding to 40 S ribosomal subunits, and promotion of mRNA binding to 40 S and 80 S ribosomes(5, 6) . Thus, eIF-1A has pleiotropic effects at different steps of the initiation process. Since the factors purified from wheat germ and rabbit reticulocytes are functionally interchangeable in vitro(7) , their active domains must be strongly conserved.

The cloning of the human eIF-1A cDNA creates an opportunity to study the factor in greater detail. We report here the overexpression of recombinant eIF-1A in Escherichia coli and the purification of large amounts of active protein. Antibodies prepared in rabbits were used to quantitate cellular levels. We also describe studies of the structure and translational efficiency of eIF-1A mRNA in vivo and the effects of overexpression in transiently transfected cells.

MATERIALS AND METHODS

Overproduction of Recombinant eIF-1A (rc-eIF-1A) in E. coli

Figure 5: Four independent cDNA clones encoding eIF-1A. Sequence relationships are depicted of four independent eIF-1A cDNA clones isolated as described previously(3) . The solidhorizontallines represent cDNA sequences identical to one or more of the other clones; the hatchedboxes represent the eIF-1A coding regions that are aligned vertically. Restriction enzyme sites are identified above each cDNA. The horizontaldashedline on the left side of clone II represents cDNA sequence not shared by any other clone. kb, kilobase pairs.

Purification of rc-eIF-1A

AUG-directed Synthesis of Methionylpuromycin

Preparation of Polyclonal Antibody against rc-eIF-1A

Affinity Purification of Antibody

Transient Transfection and Labeling of COS Cells

RESULTS

Overproduction and Purification of eIF-1A from E. coli

Figure 1: Expression, purification, and activity of rc-eIF-1A. A, recombinant eIF-1A was expressed in E. coli from pT7-7-1A as described under ``Materials and Methods.'' E. coli lysates (4 µl) were fractionated by 15% SDS-PAGE(22) ; shown is a computer scan of the Coomassie Blue-stained gel transformed with the vector pT7-7 (lanes 2 and 3) or with pT7-7-1A (lanes 4 and 5). In lanes 3 and 5, cells were treated with rifampicin following heat induction. Molecular mass markers are shown in lane1 and are identified in kilodaltons on the left. The migration position of HeLa eIF-1A is shown by an arrow on the right. B, rc-eIF-1A was purified as described under ``Materials and Methods'' and analyzed by 15% SDS-PAGE as described for A. Shown is a computer scan of a Coomassie Blue-stained gel lane containing 23 µg of protein. The band at 68 kDa is a staining artifact present in other lanes lacking eIF-1A (not shown). Molecular mass markers are shown on the right in kilodaltons. The computer scans were generated with a ScanJet IIcx scanner and the Diskscan program (Hewlett-Packard Co.). C, shown is the activity of rc-eIF-1A. The activities of rc-eIF-1A and eIF-1A purified from HeLa cells were measured by the methionylpuromycin synthesis assay as described under ``Materials and Methods.'' Activities are reported as -fold stimulation, where 1-fold represents 1594 cpm [H]methionylpuromycin formed.

An in vitro methionylpuromycin synthesis assay was used to determine whether or not rc-eIF-1A possesses initiation factor activity. The assay measures the formation of methionylpuromycin on 80 S ribosomes in the presence of AUG and five purified initiation factors (5) : eIF-1A, eIF-2, eIF-3, eIF-5, and eIF-5A. The activities of rc-eIF-1A and eIF-1A purified from HeLa cells were determined as described under ``Materials and Methods.'' rc-eIF-1A and native eIF-1A have essentially identical specific activities in this assay (Fig. 1C).

eIF-1A Is an RNA-binding Protein

Figure 2: Northwestern blot analysis. Purified rc-eIF-1A (0.68 and 1.7 µg (first and secondlanes, respectively)) and eIF-3 (2 µg (third lane)) were subjected to 15% SDS-PAGE and electrotransferred to a nitrocellulose membrane. The membrane was treated with binding buffer (20 mM HEPES-KOH, 75 mM KOAc, 2 mM Mg(OAc), 1 mM EDTA, 1 mM dithiothreitol, and 0.2% (w/v) CHAPS, pH 7.5) for 20 min and then was incubated with a solution containing binding buffer, P-labeled Xenopus -globin mRNA, and 1 mg/ml calf liver tRNA. The P-labeled probe was prepared in vitro with Xenopus -globin cDNA cloned in a pSP64 vector (23) and cleaved with BamHI, [-P]GTP (50 µCi, 10 cpm/pmol), and the MEGAscript SP6 transcription kit (Ambion Inc.) based on the manufacturer's instructions. The transcript consists of the first 354 nucleotides of Xenopus -globin mRNA including the AUG initiator codon and was purified through a size column (Stratagene Nuctrap push column), followed by precipitation with ethanol. Following incubation of the membrane and probe for 10 min, the membrane was washed three times for 5 min each with binding buffer and was subjected to autoradiography. The position of eIF-1A (labeled 1A on the right) was localized by immunoblotting of the same membrane. The p66 subunit of eIF-3 is labeled on the right; molecular mass markers are shown on the left in kilodaltons.

Cellular Level of Human eIF-1A

Figure 3: Quantitation of eIF-1A level in HeLa cells. A, shown are results from Western blot analyses. HeLa cells (1 10) were grown in flasks in RPMI 1640 medium (Hyclone Laboratories) supplemented with 10% calf serum. Cells were collected by centrifugation, washed twice with ice-cold PBS, and lysed in 1.72 ml of lysis buffer (20 mM HEPES-KOH, 10 mM MgCl, 1 mM dithiothreitol, and 0.5% Nonidet P-40, pH 7.4) with 14 strokes of a Dounce homogenizer. Cell lysates were clarified by centrifugation, and protein concentration was determined by the Bradford assay (Bio-Rad). The indicated amounts of purified rc-eIF-1A and HeLa lysate proteins were subjected to 15% SDS-PAGE (22) and electrotransferred (40 min at 300 V) to an Immobilon polyvinylidene difluoride membrane (Millipore) in CAPS transfer buffer (10 mM CAPS, 10% methanol, pH 11). Blots were blocked in PBS, 0.5% nonfat dry milk solution for 1 h; incubated with affinity-purified eIF-1A antibodies (1:500 dilution) for 16 h and then with alkaline phosphatase-conjugated anti-rabbit IgG antibodies (1:10,000 dilution); and developed with the chemiluminescence detection system (Tropix Inc.). Shown is a computer scan (see Fig. 1) of the developed film. B, band intensities from A were quantitated with a densitometer (Molecular Dynamics Model 300A) and are plotted against nanograms of rc-eIF-1A protein to generate a standard curve. The levels of eIF-1A in the lysates were calculated from band intensities matched to the standard curve as shown. Boxesa-d represent 3, 6, 9, and 12 µl of HeLa lysate at 4.3 mg/ml protein. C, the eIF-1A amounts determined for each of the lysates are plotted against the amount of lysate analyzed. The resulting straightline was obtained by a least-squares fit of the data points. The slope of the line corresponds to an eIF-1A level of 0.010% of total protein, or to 6 10 molecules/cell, based on 154 pg of protein/cell (11) and a mass of 16.4 kDa.

The affinity-purified antibodies were used in Western blot analyses to quantitate the level of eIF-1A in HeLa cells (Fig. 3). The cellular level of eIF-1A was estimated by comparing the intensities of the bands from crude lysates with the intensities obtained from known amounts of purified protein. Based on the slope of the line in Fig. 3C, eIF-1A represents 0.010% of total protein, which corresponds to 6 10 molecules/cell (see legend to Fig. 3for calculations). Since the concentration of ribosomes in HeLa cells is 3 10 ribosomes/cell(11) , the factor/ribosome ratio is 0.2. This ratio is somewhat lower than those reported previously for eIF-2, eIF-3, and eIF-4B (0.5-0.7)(11) . Therefore, eIF-1A may be a limiting initiation factor since its molar level is comparable to that of eIF-4(12) .

Expression of Human eIF-1A in COS-1 Cells

Figure 4: Transient transfections of COS-1 cells. A, the expression vector pMT2-1A. Construction of the plasmid is described under ``Materials and Methods.'' Elements in the vector are the bacterial replication origin (ColEIOri), -lactamase gene (BLA), mammalian replication origin (SV40ori), adenovirus major late promoter (Ad MLP), adenovirus tripartite leader (TPL and Intron), eIF-1A coding region, DHFR coding region, SV40 polyadenylation signal, and adenovirus virus-associated RNAs I and II (VAI and II). B, detection of overproduced eIF-1A by Western immunoblotting. Shown are immunoblots of COS cells transiently transfected with pMT2-1A and/or pMT2 as indicated. Each gel lane contains equal amounts of hot trichloroacetic acid-precipitable counts from the cells labeled with [S]methionine as described under ``Materials and Methods.'' Shown is a scan of the blot stained with the chromogenic substrates for alkaline phosphatase, nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Molecular mass markers are shown on the left in kilodaltons; the migration position of HeLa eIF-1A is shown by an arrow on the right. C, the same blot analyzed in B subjected to autoradiography, followed by scanning in a Molecular Imager System (Bio-Rad). Molecular mass markers are shown on the left in kilodaltons; the migration position of DHFR is shown by an arrowhead on the right. The band corresponding to eIF-1A is barely detectable and is marked by an arrow on the right. D, Northern blot analysis. RNA was isolated from parallel transfection plates and analyzed by Northern blot hybridization with a DHFR probe as described in the legend to Fig. 6. The probe detects both DHFR mRNA (identified by an arrowhead) and the dicistronic mRNA that encodes eIF-1A (identified by an arrow).

Figure 6: Northern blot hybridization of eIF-1A mRNA. Total RNA was prepared from exponentially growing HeLa cells as described(24) , and poly(A) and poly(A) RNAs were prepared from total RNA using an oligo(dT)-cellulose resin (Promega) according to the instruction manual. Each RNA sample was electrophoretically separated on a 1.2% formaldehyde-agarose gel and blotted onto a Hybond N membrane (Amersham Corp.) by capillary action. Hybridization was carried out with a random-primed, 0.7-kilobase pair radiolabeled SphI-XhoI DNA fragment from eIF-1A cDNA clone I (10 cpm/ml) in 5 SSC, 5 Denhardt's reagent, 0.5% SDS, and 50% formamide at 42 °C for 16 h. The membrane was washed under increasing stringency with a final wash at 55 °C in 0.1 SSC, 0.1% SDS. The blot was exposed to Kodak X-Omat AR film at -70 °C for 16 h. Shown is a computer scan of the autoradiograph, with size markers indicated in kilobases (kb) on the left and the eIF-1A mRNA labeled 1.9 kb on the right. First lane, 15 µg of total RNA; secondlane, 5 µg of poly(A) RNA; thirdlane, 5 µg of poly(A) RNA.

Characterization of eIF-1A mRNA Structure and Translational Efficiency

The 5`-leader sequence of cDNA clone II (Fig. 5) uniquely contains 214 residues at the 5`-end in addition to the 187 nucleotides shared with the 5`-UTR of clone I. To determine whether or not this extended cDNA corresponds to true eIF-1A mRNA sequences, the cDNA from clone II was used for S1 nuclease mapping of HeLa mRNA. eIF-1A mRNA protects only a partial fragment (133 nucleotides) of a 347-bp radiolabeled probe (Fig. 7A), indicating that only the 187 nucleotides of the 5`-UTR shared by clones I and II are present in eIF-1A mRNA. Primer extension was then used to identify the transcription start site(s) of eIF-1A mRNA. A single major extended cDNA product is seen (Fig. 7B); its length (139 nucleotides) indicates that the mRNA ends 187 bp upstream from the AUG start codon. These results suggest that two of the cloned cDNAs (I and III) are nearly full-length at their 5`-termini and that there is only one major transcription start site for the eIF-1A gene. However, a more precise definition and characterization of the 5`-terminus of eIF-1A mRNA requires the cloning of the corresponding gene(s).

Figure 7: Mapping the 5`-terminus of eIF-1A mRNA. A, S1 nuclease mapping of the 5`-terminus. The 347-bp EcoRI-SacII fragment from clone II (see Fig. 5; the probe extends from positions -54 to -400) was gel-purified, radiolabeled at its 5`-termini with [-P]ATP (ICN Biomedicals, Inc.) and T4 polynucleotide kinase, and heat-denatured. Hybridization was carried out at 52 °C for 6 h with E. coli tRNA (20 µg), the radiolabeled probe (2 10 cpm), and total HeLa RNA (0, 20, 40, 60, and 80 µg (lanes 2-6, respectively)) that had been heat-denatured at 75 °C for 10 min. Digestion reactions were incubated for 30 min at 37 °C with 6 µl of S1 nuclease (400 units/µl) in the presence of 20 µg of single-stranded DNA. Reactions were fractionated by electrophoresis on a 6% polyacrylamide gel in 8 M urea. The firstfourlanes are sequencing reactions used as size markers; the nucleotide (n.t.) lengths of two bands are identified on the right. Lanes1 and 7 show the undigested probe. Following exposure to Kodak X-Omat film, the film was computer-scanned. B, primer extension on eIF-1A mRNA. A 39-mer synthetic oligonucleotide primer (P3, 5`-AAGCTTCGGCGGCTGCTGCTCCGAGGGGCGACACGAGGG-3` (underlined region is the HindIII restriction enzyme site)), which is complementary to nucleotides -54 to -87 of cDNA clone I, was radiolabeled at its 5`-terminus with [-P]ATP and T4 polynucleotide kinase. Hybridization was carried out by mixing the purified P-labeled primer with HeLa poly(A) RNA (0.7, 1.4, 2.1, 2.8, and 3.5 µg (lanes 2-6, respectively)) and heat denaturing at 70 °C for 5 min with slow cooling to room temperature. The primer was extended in a reaction mixture containing 4 mM MgCl, 2.5 mM deoxynucleoside triphosphates, 25 units of RNasin, 4 mM dithiothreitol, and 100 units of Superscript reverse transcriptase (Life Technologies, Inc.) in a total volume of 25 µl at 42 °C for 1 h. The cDNA was then phenol-extracted, precipitated with ethanol, and subjected to fractionation as described for A. A set of sequencing reactions in the firstfourlanes was run as size markers. The size of the primer-extended band is shown on the left in nucleotides.

The 5`-UTR of eIF-1A mRNA is unusual in two respects: it is longer than most leaders; and it is very prone to possess secondary structure, containing 72% G + C nucleotides. Although the 5`-UTR contains no upstream AUG codon, the G/C-rich aspect leads one to predict that the mRNA will be inefficiently translated(18) . We examined eIF-1A mRNA efficiency by determining the number of ribosomes present in eIF-1A polysomes by measuring the polysome size by Northern blot hybridization. As shown in Fig. 8, eIF-1A mRNA in exponentially growing HeLa cells is found mainly in polysomes containing about five ribosomes (fractions 7 and 8), and little mRNA is detected in the ribonucleoprotein region (fractions 2 and 3). The polysome size corresponds to 85 nucleotides of coding sequence/ribosome, which is a ribosome density somewhat greater than that found on globin mRNA in rabbit reticulocytes. Reprobing the blot with an eIF-2 cDNA probe detects eIF-2 mRNA on polysomes containing at least 11-12 ribosomes, as previously reported(19) . eIF-2 mRNA is a very efficiently translated mRNA. Since the number of ribosomes on eIF-1A mRNA is about half that for eIF-2 and its coding length also is about half, the results indicate that eIF-1A mRNA translation also is very efficient, assuming that elongation/termination rates are comparable.

Figure 8: Analysis of eIF-1A polysome size. Freshly serum-fed, exponentially growing HeLa cells (2 10) were collected by centrifugation, washed twice with ice-cold PBS, and lysed with a Dounce homogenizer in 2 ml of lysis buffer (20 mM HEPES-KOH, 100 mM KCl, 10 mM MgCl, 10 µg/ml cycloheximide, 1 mM dithiothreitol, and 0.5% Nonidet P-40, pH 6.8). The lysate was clarified by centrifugation for 10 min at 8000 rpm (Sorvall SA-600), and 36 A units were applied to a linear 15-45% (w/v) sucrose gradient in detergent-free lysis buffer and centrifuged at 4 °C in a Beckman SW 40 rotor for 1 h at 38,000 rpm. Gradients were fractionated by bottom puncture and upward displacement using an Isco gradient fractionator and were scanned for absorbance at 254 nm with an Isco UV monitor (thinline). Thirteen fractions (750 µl each) from the gradient were collected, and RNA was extracted with phenol/chloroform/isoamyl alcohol, ethanol-precipitated, and analyzed by Northern blotting as described in the legend to Fig. 6. The radiolabeled probes (1 10 cpm/ml; specific activity = 1 10 cpm/µg) are the 0.7-kilobase pair eIF-1A cDNA fragment from Fig. 6and a 1.4-kilobase pair EcoRI eIF-2 cDNA fragment(19) . The relative band intensities were quantitated on a Molecular Imager System (Bio-Rad) and are plotted in arbitrary units. -, eIF-1A; - - -, eIF-2. Sedimentation is from left to right; polysomes are found in fractions 4-13.

DISCUSSION

eIF-1A functions in the early steps of protein synthesis by promoting the dissociation of 80 S ribosomes into subunits and by stabilizing the binding of the ternary complex comprising eIF-2, Met-tRNA, and GTP to 40 S ribosomal subunits. Its function in these partial initiation reactions parallels that of eIF-3 and suggests that eIF-1A also acts stoichiometrically on the ribosome. To better characterize the role of eIF-1A in protein synthesis, its cDNA was expressed in E. coli, and highly purified recombinant protein was isolated. Recombinant eIF-1A is active in vitro in the methionylpuromycin synthesis assay and displays the same molecular mass as the protein isolated from HeLa cells. Antibodies to rc-eIF-1A were prepared in rabbits and used to quantitate eIF-1A levels by Western immunoblotting. The level of eIF-1A in HeLa cell crude lysates is 0.2 molecules/ribosomes, i.e. in the micromolar range. This is about three times lower than the levels found for eIF-2, eIF-3, and a number of other initiation factors in HeLa cells(11) . Since initiation occurs on native 40 S ribosomal subunits and these usually represent <10-20% of total ribosomes in exponentially growing cells, eIF-1A is equal to or in slight excess of initiating 40 S ribosomal subunits. It follows that eIF-1A may not be limiting for protein synthesis under most conditions. Consistent with this suggestion is the finding that overexpression of the factor in transiently transfected COS cells raises the factor level 20-fold, but does not significantly affect the rate of protein synthesis.

eIF-1A is found primarily in the high salt ribosomal wash subcellular fraction(20) , suggesting an association with ribosomes. However, it is not known how the factor binds to initiation complexes and whether or not it is released following completion of the initiation phase. We demonstrate here that eIF-1A is a strong RNA-binding protein. The arginine- and lysine-rich regions in the N-terminal half of the molecule likely are responsible for this activity, although this has not yet been demonstrated directly. Since eIF-1A promotes the dissociation of 80 S ribosomes, it likely binds to one of the ribosomal subunits. Voorma and co-workers (21) have shown eIF-1A binding to 40 S preinitiation complexes containing Met-tRNA, eIF-2, and eIF-3. Its binding is promoted by eIF-1 and somewhat by the presence of mRNA. eIF-1A binding to such ribosomal complexes appears to be quite labile(21) , and there is no direct evidence that the factor binds to 40 S subunits in the absence of other translational components. We analyzed HeLa cell lysates fractionated by sucrose density gradient centrifugation for the presence of eIF-1A and found that the protein is present primarily toward the top of the gradient, but is detectable in the 40 S region and at even less abundance up to 80 S (data not shown). Whereas these results suggest a weak binding interaction with 40 S and 80 S ribosomes and perhaps with polysomes, the analyses have not been successful in demonstrating discrete binding complexes.

eIF-1A appears to be expressed from a single size class ofmRNA. The mRNA is unusual in possessing a long 5`-untrans-lated leader. Both S1 nuclease protection and primer extension analyses identify a 200-nucleotide leader upstream from the AUG initiator codon. However, we cannot rule out the possibility that still additional residues are present in the leader. There is no upstream AUG sequence, but the leader is unusually rich in G and C residues (72%). Long G/C-rich leaders are found in mRNAs that tend to be translated inefficiently(18) , due presumably to stable secondary structures. Indeed, the 5`-leader of eIF-1A mRNA may form a structure that possesses a stability of -71 kcal/mol (Fig. 9). Paradoxically, eIF-1A mRNA in HeLa cells is present in quite large polysomes, suggesting a high rate of initiation (assuming that the elongation/termination rates reflect the bulk of translation). In contrast, in vitro synthesized RNAs possessing the entire 5`-leader are translated poorly in a reticulocyte lysate, whereas the coding region lacking the 5`-leader is translated well in vitro (data not shown). Further studies of the mechanism of initiation on eIF-1A mRNA are needed to explain how in vivo translation is so efficient with this mRNA.

Figure 9: Secondary structure model of the 5`-UTR of eIF-1A mRNA. A model of the lowest energy secondary structure for the 200-nucleotide 5`-UTR of eIF-1A mRNA (plus an EcoRI site at the 5`-terminus) was generated by the program of Zuker and Steigler (25) on a Macintosh Quadra 650 computer. The calculated stability of this hypothetical structure is -71.5 kcal/mol.

FOOTNOTES

*

The work was supported by National Institutes of Health Grant GM22135 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Dept. of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 70 Massachusetts Ave., Cambridge, MA 02139.

¶

To whom correspondence should be addressed. Tel.: 916-752-3235; Fax: 916-752-3516; JWHERSHEY{at}ucdavis.edu.

()

The abbreviations used are: eIFs, eukaryotic initiation factors; rc-eIF-1A, recombinant eukaryotic initiation factor 1A; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); DHFR, dihydrofolate reductase; UTR, untranslated region; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]]-1-propanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

ACKNOWLEDGEMENTS

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				M. Bushell, W. Wood, G. Carpenter, V. M. Pain, S. J. Morley, and M. J. Clemens Disruption of the Interaction of Mammalian Protein Synthesis Eukaryotic Initiation Factor 4B with the Poly(A)-binding Protein by Caspase- and Viral Protease-mediated Cleavages J. Biol. Chem., June 22, 2001; 276(26): 23922 - 23928. [Abstract] [Full Text] [PDF]

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