The Gene Encoding the Elongation Factor P Protein Is Essential for Viability and Is Required for Protein Synthesis*

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (Kan R ) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-Kan R gene in the chromosome grew at 33 °C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 °C. Incorporation of various isotopesin vivo suggests that translation is impaired in theefp mutant at 44 °C. At 44 °C, mutant cells are severely defective in peptide-bond formation. We conclude that theefp gene is essential for cell viability and is required for protein synthesis.

The most important catalytic function of the ribosome is the synthesis of peptide bonds. A variety of approaches have been used to deduce the components that comprise this catalytic center. The results of in vitro reconstitution studies, photochemical cross-linking of substrates, and mutagenesis of conditionally lethal or antibiotic-resistant phenotypes have implicated domain V of the 23 S rRNA as well as proteins L2, L3, and L4 as the minimum components of this active center (1)(2)(3)(4)(5)(6).
A surprising finding is that the in vitro reconstituted peptidyltransferase cannot condense all aminoacyl-tRNA template combinations (7). This anomaly is reflected in the fact there is a subsite on domain V of 23 S rRNA that is specific for hydrophobic amino acids (2). In retrospect, it has been known for more than two decades that puromycin, which is one of the most common substrate analogues used to study this reaction, has a special three-dimensional structure (a U shape) that favors peptide-bond synthesis (8). Substitution of the aromatic residue of puromycin by that of other amino acids distorts this structure and drastically impairs peptide-bond synthesis (9). This specificity is reflected in the 50 S catalyzed "fragment" reaction that has been used to deduce the components of the peptidyltransferase catalytic center.
Reconstitution studies as well as photoaffinity labeling experiments indicate that several proteins of the 50 S particle enhance peptide-bond synthesis. The assembled peptidyltransferase in the 70 S ribosome catalyzes peptide bonds at a higher rate than does the peptidyltransferase of the 50 S subunit, but does not efficiently condense nonaromatic amino acids (7,10). In addressing this issue, we asked whether proteins that stimulate reconstitution of translation from homogeneous translation factors enhance the condensation of several amino acids. A soluble protein, EFP, 1 indeed stimulates the rate of peptidebond synthesis on 70 S ribosomes and, together with components of the 70 S, may be involved in restoring the ability to condense several amino acids to the peptidyltransferase (10 -12).
EFP stimulates peptide-bond synthesis by 70 S ribosomes between fMet-tRNA f Met and analogues of various aminoacyl-tRNAs. For example, the KЈ for cytidyl(3Ј-5Ј)-[2Ј(3Ј)-O-L-aminoacyladenosine (CA)-Gly is enhanced 50-fold, whereas that for CA-Phe is essentially unaltered by EFP (10). EFP may modulate the efficiency of protein synthesis by controlling the rate of synthesis of certain peptide bonds. There are 800 -900 molecules of EFP per E. coli, or about 0.1 to 0.2 copy per ribosome, suggesting that EFP may function catalytically in the cell (13).
The requirements for peptide-bond and ester-bond formation stimulated by EFP have been studied with fMet-tRNA f Met bound to 30 S subunits and native or reconstituted 50 S subunits. EFP functions in both peptide and ester-bond synthesis promoted by the peptidyltransferase (12,14,15). The 50 S particle's L16 (or its N-terminal fragment) are required for the EFP-mediated synthesis of peptide bonds, whereas L11, L15, and L7/L12 are not required in this reaction, suggesting that EFP may function at a different ribosomal site than most other translation factors (15).
To obtain the sequence of EFP as well as to examine whether it represents an essential cellular function, we have cloned and sequenced the gene encoding this protein (16). The efp gene sequence is unique and is represented only once in the E. coli chromosome. The EFP protein has been overexpressed, purified to homogeneity and crystallized (17).
To learn whether the gene encoding EFP is essential, we introduced a kanamycin marker in the early coding region of the E. coli efp gene and cloned the interrupted gene into the pMAK705 plasmid. pMAK705 contains a temperature-sensitive origin of replication. A homologous recombination proce-* This work was supported in part by the Natural Science and Engineering Council of Canada, the J. P. Bickell Foundation, and the NATO Foundation for a travel award which was used to complete this study. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  dure was used to replace the efp gene in the E. coli chromosome with the mutant copy of the gene. We show that integration of the efp-Kan R gene into the E. coli chromosome results in a lethal phenotype indicating that the efp gene is indeed essential for growth.

MATERIALS AND METHODS
Strains and DNAs-pMAK705 (5.6 kb) was a gift from Drs. Sidney R. Kushner and Valerie Maples, University of Georgia. pBluescript (2.96 kb) and XL1-blue were from Stratagene. The Kan R Genblock (1.28 kb) fragment was obtained from Pharmacia. The host strain, E. coli JM101 is recA ϩ . The temperature-sensitive cloning vector, plasmid pMAK705, is a mutant isolate of pSC101 (called pH01) (18). pMAK705 contains the temperature-sensitive replicon from pH01, the gene for chloramphenicol resistance (cm r ) from pBR322, the M13mp19 polylinker and the coding sequence for the ␣-peptide of ␤-galactosidase, which allows for the blue-white colony color selection when using selective media (IPTG, X-Gal) to identify genes that have been inserted into the plasmid.
Plasmid and Genomic DNA Isolation-Plasmid DNA was isolated by the method of Birnboim and Doly (19). To isolate genomic DNA, E. coli JM101 cells (1 ϫ 10 8 /ml) were washed with 1 ϫ SSC twice and were suspended in 1 ϫ SSC supplemented with 27% sucrose. After incubation with lysozyme (10 mg/25 ml) at 37°C for 20 min, SDS was added to a final concentration of 1% and the reaction was incubated at 60°C for 10 min. Pronase (1 mg/ml final) was then added and the solution was incubated at 37°C for 7 h. After the addition of an equal volume of phenol, the mixture was rotated gently (20 -30 min), then spun at 3000 rpm for 10 min. After two phenol extractions, DNA was dialyzed three times with 1 ϫ SCC. The buffer was changed every 12 h. Plasmid DNA was removed from chromosomal DNA by agarose gel electrophoresis. The chromosomal DNA was isolated by electroelution.
PCR and Sequence Analysis of Amplified DNA-High performance liquid chromatography-purified oligonucleotide primers were provided by the Hospital for Sick Children Biotechnology Service Center (University of Toronto). Oligo EFP 1 is 5Ј-CGTATTCACCAGAGCGGGTA-TC, which is complementary to a 20-bp sequence of the efp coding region. Oligo EFP 2 is 5Ј-AGCAACGATTTTCGTGCTGGTC, which is the sequence beginning 15 bp downstream of the start site of the coding region. KAN 3 is 5Ј-GAGATTTTGAGACACAACGTGG, which is complementary to the sequence upstream of the N-terminal end of the kanamycin resistance (aminoglycoside 3Ј-phosphotransferase) gene. KAN 4 is 5Ј-GGTTGTAACACTGGCAGAGCAT-3Ј, which is complementary to the C-terminal end of the Kan R gene. E. coli genomic DNA (0.5 mg) was amplified in 50 ml of 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2 , 0.1% gelatin, 200 mM dATP, 200 mM dCTP, 200 mM dGTP, 200 mM dTTP. PCR was carried out using combinations of the 5Ј-end and of the 3Ј-end primers, each at 1 mM, and 1 unit of Thermus aquaticus DNA polymerase (Life Technologies, Inc.). The samples, overlain with 50 ml of mineral oil (Merck) in 0.5-ml microcentrifuge tubes, were placed in an Intelligent Heating Block (Cambio), and heated to 94°C for 3 min. The reaction was performed through 30 cycles of 1 min at 48°C, 3 min at 70°C, and 1 min at 94°C and finally incubated for 5 min at 50°C and for 20 min at 70°C. After amplification, the PCR mixtures were separated in 1% agarose gels and alkaline-blotted onto nitrocellulose membranes (Bio-Rad). The membranes were prehybridized and hybridized at 58°C in 6 ϫ SCC (1 ϫ SCC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.2% skim milk, 0.2% polyvinylpyrollidone, 1% SDS, 0.005% sodium pyrophosphate, and denatured herring sperm DNA (0.1 mg/ml). Hybridization was for 3-12 h with each DNA probe (containing up to 10 6 dpm/ml). To detect the labeled DNA, the membranes were washed briefly in 2 ϫ SSC at 58°C, and twice for 10 min in 0.1 ϫ SSC, 0.1% SDS at 65°C. The membranes were blotted dry, There are about 50 nucleotides between the EcoRI and the SacI or KpnI II sites within this region. The relevant restriction enzyme sites of the efp-Kan R and of the efp gene and their size are also shown. B, restriction enzyme patterns of two plasmid isolates derived from cells carrying the interrupted efp gene. Lanes 1 and 14 have calibrated DNA molecular size ladders. Lanes 2-4 have the EcoRI digests of M13 pBluescript and of the pMAK705 plasmids isolated from mutants 5 and 13, respectively; lanes 5-7 have the SacI/KpnI digests of the M13 pBluescript and of the pMAK705 plasmids isolated from mutants 5 and 13, respectively. Lanes 8 -10 have the KpnI digests of the M13 pBluescript and of the pMAK705 plasmids isolated from mutants 5 and 13, respectively. Lanes 11-13 have the undigested M13 pBluescript and pMAK705 plasmids isolated from mutants 5 and 13, respectively. The two upper bands in the EcoRI digests of the plasmids isolated from the mutants (lanes 3 and 4) are identical to those obtained with pMAK705 (not shown). The faint 600-bp EcoRI fragment isolated from the mutants contains the C-terminal 500 bp of the efp gene. The pMAK705 vector did not harbor the 800-bp SacI/KpnI efp fragment present in both pMAK705 plasmids isolated from the mutants. C, detection by PCR of amplification products from chromosomal DNA. The EFP 1 and EFP 2 primers were annealed to the chromosomal DNA isolated from the wild-type or from the mutants harboring the efp gene interruption and were treated as described under "Materials and Methods." The reactions were run on 1% agarose gels and were stained with ethidium bromide. The size scale on the 100-bp ladder is on the first lane. Lane 2 shows the PCR product (0.6 kb) from the wild-type strain (JM101). Lanes 3-6 show the PCR product (1.9 kb) isolated from chromosomal DNA derived from colonies 5, 11, 12, and 13 harboring the efp gene interrupted by the Kan R gene. The PCR product produced with the Kan R primers (see "Materials and Methods") corresponds to 1.3 kb (not shown). covered in Saran Wrap, and exposed to Kodak X-Omat AR film for at least 2 h, where necessary with intensification screens.
After PCR amplification, the DNA was purified by agarose gel electrophoresis. Dideoxy DNA sequencing was determined by the method of Sanger et al. (20). Sequencing was performed after hybridizing the appropriate DNA probes using DNA Sequenase (Stratagene).
Recombinant DNA Methods-Transformation of E. coli and agarose gel electrophoresis were performed as described previously (21). Restriction endonuclease enzyme digestions and ligations were carried out as outlined by the manufacturers (Life Technologies, Inc. and New England Biolabs).
Construction of an efp Gene Mutant-The N-terminal end of the efp gene has a unique EcoRI restriction site (16) that was used to insert the kanamycin resistance gene. For convenience, the efp-Kan R fragment was first ligated into the KpnI/SacI site of M13 pBluescript. After transformation of competent JM101 cells, the plasmid was isolated once again from lac Ϫ clones that were kanamycin-and ampicillin-resistant. Aliquots of the plasmids were treated with KpnI/SacI or EcoRI, and the restriction digests were analyzed electrophoretically on 1% agarose gels. The fragment carrying the efp-Kan R gene was isolated electrophoretically and was cloned into the KpnI/SacI site of plasmid pMAK705. The pMAK705 plasmid, carrying the interrupted efp gene, was transformed into JM101 cells at 33°C in LB broth. After transformation was completed, integration of the plasmid was selected by overnight growth at 44°C in LB medium in the presence of chloramphenicol (20 g/ml) and kanamycin (50 g/ml). Twenty-four white colonies (co-integrates) were selected and inoculated into 100 ml of LB broth containing chloramphenicol and kanamycin, and allowed to grow overnight at 33°C so that a second recombination event could take place resulting in the resolution of the plasmid from the chromosome. Two more cycles of growth were carried out by inoculating 100 ml of an overnight culture into 100 ml of fresh medium. Single colonies were isolated by plating dilutions of the overnight culture on LB media containing chloramphenicol, kanamycin, IPTG, and X-gal and were incubated at 33°C overnight.
To distinguish between cells carrying plasmid resolution products (i.e. plasmid free from the chromosome) from unresolved co-integrates, single colonies were tested for chloramphenicol resistance at 44°C. Colonies that are chloramphenicol-sensitive (cm s ) at 44°C should no longer contain the plasmid DNA integrated into the chromosome (22). However, no colonies that exhibited sensitivity to chloramphenicol at 44°C could be isolated from hundreds of clones that were searched.
Purification of EFP from the Mutant and from Wild-type Cells-Cells from the wild-type JM101 or from the mutant M-13 strain were grown at 33°C in L broth containing 50 g/ml of kanamycin. After cells had reached mid-log, the temperature was raised to 44°C and growth was continued for one hour. The cells were harvested and 100 g (wet weight) were broken with an equal weight of acid washed Alcoa Alumina 305. One hundred ml of 10 mM Tris, pH 7.4, 1 mM dithiothreitol, 50 mM NH 4 Cl, and 10 mM MgCl 2 (buffer A) were added as well as 2 g/ml DNase (RNase-free). The unbroken cells and debris were removed by centrifugation at 16,000 ϫ g twice in a Sorvall centrifuge. Ribosomes were isolated by centrifugation at 105,000 ϫ g for 3 h in a Beckmann ultracentrifuge. The ribosomal wash was extracted in 1 M NH 4 Cl in buffer A and was dialyzed against buffer A without NH 4 Cl. The ribosomal eluate was purified batchwise with QEA-cellulose (25 g/wet weight) eluted twice using 100 ml of 150 mM NH 4 Cl, once with 100 ml of 200 mM NH 4 Cl, and once with 100 ml of 500 mM NH 4 Cl in buffer A. EFP was assayed using f[ 35 S]Met-tRNA f Met bound to 70 S MRE600 ribosomes. The amount of f[ 35 S]Met-puromycin formed was analyzed as described previously (12).

RESULTS
The gene replacement method used to delete the efp gene was adapted from that of Hamilton et al. (22). The method makes use of a temperature-sensitive pSC101 replicon to facilitate gene replacement (18). A mutant strain is created when the gene fragment of interest is mutated i.e. by deletion, sitedirected mutagenesis or interruption by another gene, and then cloned into the replicon. This replicon, carrying the mutated gene, can then be transformed into an appropriate host. Homologous recombination can then occur between the wildtype gene on the chromosome and the homologous sequences of the mutated gene carried on the temperature-sensitive plasmid. After transformation is completed, it is possible to select for integration of the plasmid into the chromosome at 44°C. If these co-integrates are then grown at 33°C, a second recombination event takes place resulting in the resolution of the plasmid from the chromosome. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retained the original copy of the gene. This technique will establish if a gene is essential.
Evidence that the efp Gene Was Replaced by the efp-Kan R Gene in the E. coli Chromosome-Twenty-four colonies that grew at 33°C but not at 44°C in kanamycin were isolated as described under "Materials and Methods" to determine if the plasmid that was present contained the mutant copy or the chromosomal copy of the gene. The plasmids isolated from colonies that grew at 33°C in kanamycin were subjected to restriction enzyme analysis. Fig. 1A shows the position of the relevant restriction enzyme sites within the polyclonal site region of the pMAK705 plasmid harboring the efp-Kan R gene as well as the relevant restriction sites of the efp and efp-Kan R genes. For convenience the efp-Kan R gene was cloned into the SacI/KpnI site of M13 pBluescript. The SacI/KpnI fragment was excised and cloned into the corresponding SacI/KpnI site of pMAK705. pMAK705 has unique KpnI and SacI sites within this region. The kan R and efp genes, on the other hand, have no KpnI or SacI sites and the kan R gene has no EcoRI sites. The efp gene has an EcoRI site close to the 5Ј-terminus of the coding region. A second EcoRI site occurs in the pMAK705 polyclonal site. Therefore, EcoRI was used to detect the kan R gene and KpnI/SacI was used to detect the (0.8 kb) efp gene. The coding sequence of the efp gene is 0.6 kb; but the 0.8-kb region har- boring the upstream region was cloned into pMAK705. Fig. 1B shows the restriction enzyme analysis of the plasmid isolated from two typical mutant clones where the efp-Kan R gene in pMAK705 was replaced by a gene of identical size to that of efp. Plasmid DNA isolated from 23 of the 24 colonies had the same restriction pattern. The inserts exhibit the restriction enzyme pattern expected of the efp gene, further suggesting that the pMAK705 plasmid isolated from these temperature-sensitive clones now harbors the wild-type efp gene and not its interrupted version.
The presence of the mutant efp in the E. coli chromosome was verified by PCR. Total DNA was isolated from wild-type and mutant cells. PCR was then performed on each sample, using primers specific to the 3Ј-and 5Ј-regions of both the efp and the Kan R genes. A PCR fragment of the wild-type DNA primed with the probes complementary to the efp gene has the size expected (0.6 kb) of the efp gene (Fig. 1C, lane 2). However, the PCR of the chromosomal DNA from three mutant clones with the kanamycin insert showed a 1.9-kb band (Fig. 1C, lanes   4 -6), which corresponds to the efp-Kan R gene. The insertion of the kanamycin gene into the E. coli chromosone was also confirmed by the sequencing of the PCR products using primers complementary to the kanamycin gene (data not shown).
Effect of efp Gene Replacement on Protein Synthesis and on Cell Growth-If the efp gene is essential, the cells containing the efp-Kan R chromosomal copy and the wild type copy of the gene on the plasmid will appear conditionally lethal under these conditions and should be kanamycin resistant at the permissive temperature. The reason is that the mutant gene on the chromosome can no longer be complemented by the wildtype gene on the plasmid since the pMAK705 does not replicate at 44°C. Cells containing the altered chromosomal copy of the efp gene on the pMAK705 plasmid were grown at 44°C in LB broth in the absence of chloramphenicol in attempt to cure the plasmid from the cell. Indeed, mutant strains grew well at 33°C but did not grow well or at all at 44°C (Fig. 2, A and B). This suggested that the efp gene is essential for cell viability.
To verify further if the inserted efp gene on the pMAK705 were grown at 33°C. After 60 min of growth, the temperature was shifted to 44°C. B-D, DNA, RNA, and protein synthesis by wild-type and mutant strains at permissive and nonpermissive temperatures. JM101 cells or JM101 cells harboring the efp Kan R were grown at 33°C for 30 min and were shifted to 44°C for 10 min prior to the addition of the radioactive labels. B, an aliquot (0.2 ml) of the cultures was labeled with -5 Ci of [methyl-3 H]thymidine (20 Ci/mmol; NEN Life Science Products), and 0.2 M thymidine was added. Ten-l aliquots were withdrawn at the indicated times and precipitated and washed with 5% cold trichloroacetic acid prior to determining the radioactivity. C, an aliquot (0.2 ml) of the JM101 or mutant cultures was labeled at 44°C with 5 Ci of [5-3 H]uridine (20.6 Ci/mmol; Amersham Corp.). Ten-l aliquots were withdrawn and treated as in B. D, growth of the mutant was at 33°C. 10 Ci of [ 35 S]Met (1,000 Ci/mmole; Amersham Corp.) were added to 0.25-ml aliquots of the culture, and growth was continued at 33°C, or the temperature was shifted after 60 min to 44°C. Protein synthesis was measured on the 10-l aliquots at the indicated times as acid-insoluble precipitates (42). plasmid is essential for growth, we attempted to substitute this plasmid with pUC18 or M13 pBluescript which harbor a ampicillin resistance marker. JM101 is ampicillin-sensitive, but transformation of JM101 cells with these plasmids results in growth in ampicillin (data not shown). JM101 cells harboring the efp-Kan R gene and pMAK705 carrying the wild-type efp gene were transformed at 33°C with the pUC18 or M13 pBluescript plasmids with or without the efp gene insert. Transformation of JM101 cells harboring the efp-Kan R gene by M13 pBluescript or pUC18 does not result in growth at 44°C (Fig. 2,  C and D, plates 1 and 2, upper half). Cells grow very well on ampicillin at 44°C only when the 0.8-kb fragment containing the efp gene coding sequence was cloned into either M13 pBluescript or pUC18 (Fig. 2, C and D, plates 1 and 2, lower half). Thus, the presence of the efp gene in these plasmids is indeed essential for growth.
To establish whether the efp gene affects translation in vivo, we grew one of the mutant strains at 33°C in the presence of kanamycin and shifted the temperature to 44°C after the cells had entered the logarithmic phase of growth. Cells harboring the efp gene on the pMAK705 plasmid have a generation time of 30 min at 33°C, whereas cells from the wild-type JM101 strain double every 24 min. Growth of cells from the mutant begins to slow almost immediately when the temperature is shifted to 44°C. In contrast, the wild-type strain continues to grow at 44°C (Fig. 3A).
After 60 min of growth at 33°C, the JM101 cells harboring the efp-Kan R gene were shifted to 44°C and were incubated with [ 35 (Fig. 3, B and C). The [ 35 S]methionine continues to be incorporated into protein by the JM101 wild-type strain after the shift in temperature to 44°C (data not shown). JM101 cells harboring the efp-Kan R gene incorporate [ 35 S]methionine efficiently at 33°C, but incorporation of [ 35 S]methionine stops when the cultures are shifted to 44°C (Fig. 3D).
Cells from the control and mutant strain were grown at 33°C and were shifted to 44°C (see Fig. 3A). The cells were harvested and were used to partially purify the EFP protein.
This was done to remove inhibitors from the extracts. A single step of purification on QEA-Sepharose sufficed to accomplish this. As shown in Fig. 4, the ability of the extracts to stimulate peptide bond synthesis is markedly decreased when the cells are grown at 44°C to deplete them of EFP. In contrast, the control cells exhibit the expected level of fMet-puromycin synthesis. Taking the activity of EFP recovered from the wild-type strain as 100%, only 20% of the activity is recovered from the strain harboring the interrupted EFP gene. These experiments therefore show that the mutant strain is indeed defective in the EFP function and that depletion of EFP from the cell results in an abrupt cessation of growth. DISCUSSION A surprising result has been the demonstration that the genes encoding certain ribosomal proteins can be deleted from the chromosome without an apparent effect on cell viability (23). These observations have rekindled interest on whether other genes involved in translation, such as encoding certain translation factors, are essential. There is now evidence that most initiation, elongation and termination factors are required for cellular growth (24 -34). Some of these proteins, for example the termination factor RF3, may be dispensable under certain growth conditions, but not others (35). In addition to these classical translation factors are a set of proteins whose action is either stimulatory or indispensable for translation reconstituted in vitro. Of these proteins, the RRF, which is thought to be involved in disassembly of the termination complex produced by RF1 or RF2 (peptide release factors) is only stimulatory to translation in vitro yet it is encoded by an essential E. coli gene (24). Other proteins involved in the association/dissociation of ribosomes, such as the initiation factor (IF3) and the "rescue" protein, appear to be necessary for proper growth, but their action in vitro can be overcome by ionic conditions that foster the association/dissociation of ribosomal particles (36).
Of particular interest in this regard is a protein, EFP, that has been implicated in formation of the first peptide bond in vitro (10). EFP appears to be the prototype of a set of highly conserved proteins named eIF5A in eukaryotic cells (37,38). These proteins stimulate the formation of a peptide bond between the 70 or 80 S bound fMet-tRNA f Met or Met-tRNA f Met substrate and puromycin which mimics synthesis of peptide bonds. These proteins are not required for formation of initiation complexes and do not stimulate synthesis with poly(U), suggesting that they act on synthesis of the first peptide bond encoded in the native template. For the case of the Saccharomyces cerevisiae counterpart of the EFP protein, the genes are essential for growth. These mutants exhibit certain abnormalities in the pattern of their polysomes compatible with an early defect in translation. However, the eIF5A-depleted cells synthesize protein to about 60 -70% of the wild-type cells. It is not known whether leakiness of the transcribed eIF5A gene is responsible for the incomplete shut-off of protein synthesis. Alternatively, eIF5A could be selectively involved in synthesis of certain proteins that are essential for growth (39). In this context, it is of interest that the prokaryotic EFP preferentially stimulates synthesis of certain dipeptides and has substantially less affect on synthesis of other dipeptides (10). EFP does stimulate translation reconstituted with a natural mRNA but this stimulation depends on the presence of other less well characterized proteins.
Here we show by PCR and sequence analysis of the chromosomal and plasmid DNA that the interrupted efp gene replaced the wild-type copy of the chromosomal efp gene. After this gene FIG. 4. EFP activity of control, JM101, and of mutant extracts grown at 33°C and shifted to 44°C (see Fig. 3A). The EFP protein was partially purified as described under "Materials and Methods." EFP was assayed by its ability to stimulate f[ 35 S]Met-puromycin synthesis from 70S bound f[ 35 S]Met-tRNA f Met and puromycin as described in Chung et al. (12). Fractions W contain EFP activity from the QAE-Sepharose columns loaded with the extract purified from JM101. Fractions M contain residual EFP activity from the QAE-Sepharose columns loaded with extracts purified from the mutant harboring the efp gene interruption. exchange occurred, the altered cells survived only in the presence of the wild-type copy of the efp gene that was carried on the temperature-sensitive pMAK705 replicon. At restrictive temperatures, cells were unable to grow because the plasmid could not replicate. We conclude that interruption of the efp gene in the chromosome is lethal to the cell indicating that this gene is essential for growth.
A comparison of the growth and radioactive labeling properties at permissive and nonpermissive temperatures of the mutant harboring the efp gene on the chromosome and the wildtype allele in the plasmid suggests that the efp gene is required for protein synthesis in vivo. Extracts derived from mutant cells shifted during growth from permissive to nonpermissive temperatures lost 80% of their peptide-bond forming activity while the control cells remained unaltered. Since plasmids must segregate before the temperature-sensitive lesion is expressed, these results suggest that the efp gene product is essential for protein synthesis in vivo and involves a reaction directly or indirectly involving peptide-bond formation.
The bulk of the evidence indicates that the efp gene functions in an early event of translation. The sequence of this gene is conserved throughout species 2 and represents an essential function. The failure of the EFP and eIF5A proteins to be absolutely required for in vitro synthesis may depend on the conditions of the reactions, on the limitation of other proteins that may be required for reconstitution, or on the intriguing possibility that at least part of this set of gene products indeed selectively regulates the synthesis of certain essential proteins involved in control of growth (39).