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• W2066176289 abstract "Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAiand mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression ofTIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity. Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAiand mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression ofTIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity. There are five major steps involved in initiation of eukaryotic protein synthesis: dissociation of ribosomes into 40 S and 60 S subunits, binding of Met-tRNAi to the 40 S ribosomal subunit; binding of mRNA to the 40 S preinitiation complex; scanning and initiation codon recognition, and the joining of the 60 S subunit to the 40 S initiation complex (1Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar, 2Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (638) Google Scholar). The initiation phase is promoted by at least 10 soluble proteins known as eukaryotic initiation factors (eIFs). 1The abbreviations used are:eIFs, eukaryotic initiation factors; RRM, RNA recognition motif; PCR, polymerase chain reaction; kb, kilobase pair; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RNP, ribonucleoprotein. eIF3 is the largest and most complex initiation factor, comprising 10 or more subunits in mammalian cells (3Brown-Luedi M.L. Meyer L.J. Milburn S.C. Mo-Ping Yau P. Corbett S. Hershey J.W.B. Biochemistry. 1982; 21: 4202-4206Crossref PubMed Scopus (48) Google Scholar) and up to eight subunits in yeast (4Hershey J.W.B. Asano K. Naranda T. Vornlocher H.-P. Hanachi P. Merrick W.C. Biochimie (Paris). 1996; 78: 903-907Crossref PubMed Scopus (61) Google Scholar,5Naranda T. MacMillan S.E. Hershey J.W.B. J. Biol. Chem. 1994; 269: 32286-32292Abstract Full Text PDF PubMed Google Scholar). Several functions have been assigned to eIF3 in the translation initiation pathway in mammalian cells (1Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). It promotes dissociation of 80 S ribosomes into 40 S and 60 S subunits and stabilizes Met-tRNAi binding to the 40 S ribosomal subunit. eIF3 also is required for mRNA binding to 40 S and 80 S ribosomes in part through its interaction with eIF4G (6Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Crossref PubMed Scopus (472) Google Scholar) and/or eIF4B (7Méthot N. Song M.S. Sonenberg N. Mol. Cell. Biol. 1996; 16: 5328-5334Crossref PubMed Scopus (156) Google Scholar). Thus, eIF3 plays a central role in the initiation process. To assist in elucidating the function of eIF3, attention has been given to the corresponding factor in yeast, where genetic approaches are feasible. The yeast eIF3 complex was purified on the basis of its stimulation of methionylpuromycin synthesis dependent on formation of 80 S initiation complexes in an assay composed of mammalian components (5Naranda T. MacMillan S.E. Hershey J.W.B. J. Biol. Chem. 1994; 269: 32286-32292Abstract Full Text PDF PubMed Google Scholar). The active eIF3 preparation contains eight subunits with apparent molecular masses of 16, 21, 29, 33, 39, 62, 90, and 135 kDa (5Naranda T. MacMillan S.E. Hershey J.W.B. J. Biol. Chem. 1994; 269: 32286-32292Abstract Full Text PDF PubMed Google Scholar). However, further studies revealed that the 29-kDa protein is a truncated form of p39 (8Naranda T. Kainuma M. MacMillan S.E. Hershey J.W.B. Mol. Cell. Biol. 1997; 17: 145-153Crossref PubMed Scopus (47) Google Scholar) and that the p21 protein originates by partial degradation of a 110-kDa protein. 2H.-P. Vornlocher, P. Hanachi, S. Ribeiro, and J. W. B. Hershey, manuscript in preparation. A somewhat similar preparation was isolated and purified by using its stimulation of protein synthesis in lysates prepared from aprt1-1 temperature-sensitive strain of yeast. The reported molecular masses of the subunits in this preparation of putative eIF3 are 130, 80, 75, 40, and 32 kDa (9Danaie P. Wittmer B. Altmann M. Trachsel H. J. Biol. Chem. 1995; 270: 4288-4292Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The following yeast genes already have been characterized as encoding subunits of eIF3: SUI1 (p16) (10Naranda T. MacMillan S.E. Donahue T.F. Hershey J.W.B. Mol. Cell. Biol. 1996; 16: 2307-2313Crossref PubMed Scopus (61) Google Scholar), TIF34 (p39) (8Naranda T. Kainuma M. MacMillan S.E. Hershey J.W.B. Mol. Cell. Biol. 1997; 17: 145-153Crossref PubMed Scopus (47) Google Scholar), GCD10 (p62) (11Garcia-Barrio M.T. Naranda T. Vazquez de Aldana C.R. Cuesta R. Hinnebusch A.G. Hershey J.W.B. Tamame M. Genes Dev. 1995; 9: 1781-1796Crossref PubMed Scopus (59) Google Scholar), PRT1 (p90) (5Naranda T. MacMillan S.E. Hershey J.W.B. J. Biol. Chem. 1994; 269: 32286-32292Abstract Full Text PDF PubMed Google Scholar), andNIP1 (p93) (12Greenberg J.R. Phan L. Gu Z. deSilva A. Apolito C. Sherman F. Hinnebusch A.G. Goldfarb D.S. J. Biol. Chem. 1998; 273: 23485-23494Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). SUI1, encoding the smallest subunit of eIF3 (p16), was first identified genetically by recessive mutations that allow utilization of a UUG triplet as a translation initiation codon (13Yoon H.J. Donahue T.F. Mol. Cell. Biol. 1992; 12: 248-260Crossref PubMed Scopus (148) Google Scholar). TIF34 was cloned by obtaining a partial amino acid sequence of p39 and matching it to translated sequences in the data base (8Naranda T. Kainuma M. MacMillan S.E. Hershey J.W.B. Mol. Cell. Biol. 1997; 17: 145-153Crossref PubMed Scopus (47) Google Scholar) or by identifying the yeast homolog of the human TRIP1 protein (eIF3-p36) (14Verlhac M.-H. Chen R.H. Hanachi P. Hershey J.W.B. Derynck R. EMBO J. 1997; 16: 6812-6822Crossref PubMed Scopus (50) Google Scholar). The gene encoding p62 was identified through mutations that constitutively derepress the expression of GCN4 in rich medium (11Garcia-Barrio M.T. Naranda T. Vazquez de Aldana C.R. Cuesta R. Hinnebusch A.G. Hershey J.W.B. Tamame M. Genes Dev. 1995; 9: 1781-1796Crossref PubMed Scopus (59) Google Scholar). The p90 (Prt1) subunit was shown to affect Met-tRNAi binding to the 40 S ribosomal subunit (15Feinberg B. McLaughlin C.S. Moldave K. J. Biol. Chem. 1982; 257: 10846-10851Abstract Full Text PDF PubMed Google Scholar, 16Evans D.R.H. Rasmussen C. Hanic-Joyce P.J. Johnston G.C. Singer R.A. Barnes C.A. Mol. Cell. Biol. 1995; 15: 4525-4535Crossref PubMed Scopus (34) Google Scholar). The gene for p93 (NIP1) was first identified through a mutation that affects nuclear import of proteins (17Gu Z. Moerschell R.P. Sherman F. Goldfarb D.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10355-10359Crossref PubMed Scopus (29) Google Scholar). Analysis of strains carrying temperature-sensitive mutations in each of these five genes demonstrated that the proteins are required for initiation of protein synthesis in vivo(11Garcia-Barrio M.T. Naranda T. Vazquez de Aldana C.R. Cuesta R. Hinnebusch A.G. Hershey J.W.B. Tamame M. Genes Dev. 1995; 9: 1781-1796Crossref PubMed Scopus (59) Google Scholar, 12Greenberg J.R. Phan L. Gu Z. deSilva A. Apolito C. Sherman F. Hinnebusch A.G. Goldfarb D.S. J. Biol. Chem. 1998; 273: 23485-23494Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 13Yoon H.J. Donahue T.F. Mol. Cell. Biol. 1992; 12: 248-260Crossref PubMed Scopus (148) Google Scholar, 14Verlhac M.-H. Chen R.H. Hanachi P. Hershey J.W.B. Derynck R. EMBO J. 1997; 16: 6812-6822Crossref PubMed Scopus (50) Google Scholar, 15Feinberg B. McLaughlin C.S. Moldave K. J. Biol. Chem. 1982; 257: 10846-10851Abstract Full Text PDF PubMed Google Scholar). Genes encoding the p135 (TIF31) and p110 (TIF32) subunits have been identified,2 and a preliminary account of their cloning has been published (4Hershey J.W.B. Asano K. Naranda T. Vornlocher H.-P. Hanachi P. Merrick W.C. Biochimie (Paris). 1996; 78: 903-907Crossref PubMed Scopus (61) Google Scholar). To complete the cloning of the genes coding for yeast eIF3 subunits, we focused on the p33 subunit of eIF3 encoded by TIF35 (for translationinitiation factor 3, the5th subunit). TIF35 was first cloned through a two-hybrid analysis with p39 (TIF34) as bait (14Verlhac M.-H. Chen R.H. Hanachi P. Hershey J.W.B. Derynck R. EMBO J. 1997; 16: 6812-6822Crossref PubMed Scopus (50) Google Scholar). In this report, identification of the gene product as a component of eIF3 was based in large part on the work described here. During the preparation of this manuscript, reports were made identifying p33 in eIF3 complexes prepared by affinity chromatography or immunoprecipitation (18Phan L. Zhang X. Asano K. Anderson J. Vornlocher H.-P. Greenberg J.R. Goldfarb D.S. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 4935-4946Crossref PubMed Scopus (158) Google Scholar, 19Asano K. Phan L. Anderson J. Hinnebusch A.G. J. Biol. Chem. 1998; 273: 18573-18585Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). However, further characterization of p33 and its role in protein synthesis were not addressed in the reports. We describe here the cloning of TIF35, characterization of the function of p33 in protein synthesis, and analysis of its RNA recognition motif (RRM). This completes the detailed characterization of the genes encoding eight eIF3 subunits in yeast. Escherichia coli strain XL1-Blue was used for plasmid propagation. The variousSaccharomyces cerevisiae strains used in this work are based on strain W303-1A (MAT a , leu2-3,112 his3-11,15 ade2-1 trp1-1 can1-100) or its isogenic diploid, W303 (20Thomas B.J. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1352) Google Scholar). Yeast cells were grown at 30 °C in YP or synthetic (S) medium supplemented with the relevant amino acids and 2% glucose (YPD or SD) or 2% galactose (YPG or SG) as described previously (21Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. Academic Press, Inc., San Diego, CA1991Google Scholar); growth was monitored by measuring absorbance at 600 nm (A 600). Sporulation was carried out at room temperature on plates containing 0.3% potassium acetate, 0.02% raffinose, and 10 μg/ml each amino acid. Tetrad dissections and DNA transformations were carried out by standard procedures (22Struhl K. Stinchcomb D.T. Scherer S. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1035-1039Crossref PubMed Scopus (930) Google Scholar). The yeast gene encoding eIF3-p33 was tentatively identified as a homolog of the gene for mammalian eIF3-p44. The mouse eIF3-p44 sequence (34Block K.L. Vornlocher H.-P. Hershey J.W.B. J. Biol. Chem. 1998; 273: 31901-31908Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) was used to conduct a TblastN search of the entire yeast genome sequence, 3http://genome-www.stanford.edu/Saccharomyces/. and a putative protein was identified whose amino acid sequence exhibits 33.3% identity to mouse eIF3-p44. The coding sequence, together with flanking regions, was amplified from total yeast genomic DNA by PCR. The upstream (5′-CTCTTCACGATCTGCAAAAGTCCCAACATT-3′) and downstream, (5′-GCTTATGGTGGTGGTGCTTCTTATAGCGCC-3′) primers generate a single 2.1-kb DNA fragment. The fragment was gel-purified and subcloned into pNoTA (5 Prime → 3 Prime, Inc., Boulder, CO ) to create pNo-TIF35. Sequencing confirmed that the 2.1-kb insert contains an 825-base pair open reading frame (ORF) with 607 and 674 base pairs of DNA flanking the 5′- and 3′-ends, respectively. To disrupt the TIF35 gene, pNo-TIF35 was digested withBsaAI and BsmI to remove 91% of theTIF35 coding region, and a 1.7-kb BamHI DNA fragment containing HIS3 was inserted to generate pNoTAtif35::HIS3. The upstream and downstream cloning primers described above were used to generate a 3.1-kbtif35::HIS3 PCR fragment, which was transformed into the diploid yeast strain W303 to create a one-step gene deletion/disruption (23Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2030) Google Scholar). One of the stable His+transformants (PH33D-7) was selected, and the disruption of one of theTIF35 genes was confirmed by Southern blot analysis (data not shown). pRS316-TIF35 was constructed by digesting pNo-TIF35 with BamHI and subcloning the resulting fragment into BamHI-cleaved pRS316 (American Type Culture Collection). p415Gal1-NH33 is a CEN4 LEU2 plasmid that allows expression of N-terminal His6-tagged p33 under the control of the GAL1 promoter. For its construction, the p33 coding sequence was PCR-amplified from pNo-TIF35 using 5′-CCCGGATCCGCCATGGGTAGAGGTTCTCACCATCACCATCACCATATGAGTGAACATATTCTGTGCATCTA-3′ (tagged with BamHI and NcoI sites (underlined); the bases corresponding to the initiation codon of wild-typeTIF35 are in boldface) and 5′-CCCGTCGACCTCGAGCATATTCTGTGCATCTA-3′ (tagged with SalI and XhoI sites (underlined); the bases corresponding to the stop codon are in boldface). The resulting 0.9-kb DNA fragment was subcloned into pNoTA and sequenced, yielding pNo-NH33 (NH represents N terminus tagged with His6). To construct plasmids p415GalL-NH33 and p415GalS-NH33, pNo-NH33 was digested withBamHI and SalI, and the 0.9-kb fragment was subcloned into the BamHI/SalI sites of p415GalL and p415GalS, respectively (24Mumberg D. Maller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (803) Google Scholar). p415p33ΔC (kindly provided by M.-H. Verlhac, University of California, San Francisco) (14Verlhac M.-H. Chen R.H. Hanachi P. Hershey J.W.B. Derynck R. EMBO J. 1997; 16: 6812-6822Crossref PubMed Scopus (50) Google Scholar) is identical to p415Gal1-NH33, except that the encoded p33 lacks the C-terminal 71 amino acids. To express a recombinant form of His-tagged p33 in E. coli, pET-NH33 was constructed by inserting theNcoI/XhoI fragment from pNo-NH33 into the corresponding sites in pET28c (Novagen). To generate pET-NH33ΔC, p415p33ΔC was digested with BamHI and SalI, and the 0.7-kb fragment was subcloned into pET28c digested withBamHI/SalI. Strain PH33D-7 was transformed with p415GalS-NH33, p415GalL-NH33, or p415p33ΔC, and transformants were selected on SD-His-Leu plates. The resulting transformants were sporulated, and their asci were dissected. Two, three, and four viable spores were obtained on YPG plates and were streaked on SG-His-Leu plates to identify TIF35-disrupted cells carrying p415GalS-NH33, p415GalL-NH33, and p415p33ΔC. The corresponding haploid strains were named PHS33, PHL33, and PH133, respectively. Rabbit antiserum against yeast eIF3 has been described previously (5Naranda T. MacMillan S.E. Hershey J.W.B. J. Biol. Chem. 1994; 269: 32286-32292Abstract Full Text PDF PubMed Google Scholar). Rabbit anti-Prt1 antibody was a gift from A. G. Hinnebusch (National Institutes of Health), and affinity-purified rabbit anti-p39 antibody (14Verlhac M.-H. Chen R.H. Hanachi P. Hershey J.W.B. Derynck R. EMBO J. 1997; 16: 6812-6822Crossref PubMed Scopus (50) Google Scholar) was kindly provided by M.-H. Verlhac. To obtain anti-p33 antiserum, rabbit antibodies were raised against purified His6-p33 (BAbCo). For affinity-purified anti-recombinant p33 antibodies, His6-p33 was overexpressed from pET-NH33 in E. coli BL21(DE3), partially purified by Ni2+ affinity chromatography (Novagen), and fractionated by SDS-PAGE, followed by transfer to a polyvinylidene difluoride membrane (Millipore Corp.). The anti-p33 antiserum was incubated with a piece of the membrane containing His6-p33, and antibodies bound to His6-p33 were eluted with 0.2 ml of low-pH buffer (0.2 m glycine HCl and 1 mm EGTA, pH 2.5). The eluate was quickly neutralized with 0.2 ml of 100 mm Tris-HCl, pH 8.8; diluted with 1 volume of Blotto (0.5% (w/v) nonfat dry milk in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.075% (v/v) Tween 20); and stored frozen at −80° C. A second batch of affinity-purified anti-p33 antibodies was prepared similarly, but from the anti-eIF3 antiserum, and was used as indicated in the figure legends. Anti-p135 and anti-p110 antibodies were affinity-purified against recombinant His6-tagged p135 and p110 as described elsewhere.2 Strains PHS33 and W303-1A were grown in YPG medium at 30° C to early log phase and shifted into YPD medium. Five, nine, and twelve hours after the shift to glucose, cycloheximide was added to a final concentration of 100 μg/ml, followed by quick cooling of the cultures on ice. The cells were harvested by centrifugation and washed with buffer A (10 mm Tris-HCl, pH 7.4, 100 mm KCl, 10 mm MgCl2, and 1 mm dithiothreitol) plus 100 μg/ml cycloheximide. Cells were broken by vortexing with glass beads in lysis buffer (20 mm HEPES, pH 7.5, 5 mm MgCl2, 150 mm KCl, 5% (v/v) glycerol, and 1× CompleteTM protease inhibitors (Boehringer Mannheim)), and cell lysates were clarified by centrifugation at 20,000 × g for 10 min at 4° C. Aliquots (10 A 260 units) from each extract were fractionated on 15–45% sucrose gradients in buffer A by centrifugation at 38,000 rpm in a Beckman SW 40 rotor for 2.25 h at 4° C. The gradients were analyzed by upward displacement, andA 254 profiles were obtained with a density gradient fractionator and UV monitor (Isco Model 185). Strains W303-1A and PHS33 were grown overnight in SG complete minus methionine medium. Cells were harvested, washed in water, and resuspended in either SG complete minus methionine or SD complete minus methionine medium at a density of 0.05 A 600. During incubation at 30° C, cells corresponding to 1 A 600 unit were withdrawn at the indicated time points, harvested, washed in buffer A, and resuspended in 300 μl of the same medium containing 100 μCi of [35S]methionine (1 × 106Ci/mol). The cells were incubated at 30° C for 5 min, followed by addition of 1 ml of a “stop buffer” containing 1.2 mg/ml nonradioactive methionine and 0.1 mg/ml cycloheximide in lysis buffer. Cells were disrupted with glass beads, and proteins from the cleared lysate were precipitated with 10% trichloroacetic acid. The pellet was washed with acetone and dissolved in 200 μl of 1% SDS. The incorporation of [35S]methionine into total protein was determined by counting radioactivity in an aliquot of the SDS extract. The protein concentration of the SDS extract was determined by the micro-BCA protein assay reagent kit (Pierce) as described by the manufacturer. The rate of protein synthesis is expressed as cpm × min−1 × μg of protein−1. Cells harvested at an A 600 of 0.9–1 were disrupted with glass beads in lysis buffer by eight 30-s pulses in a Bead Beater (BioSpec Products, Inc.). The lysate was centrifuged for 15 min at 12,000 × g at 4° C, and the supernatant was centrifuged at 65,000 rpm in a Beckman TL100.4 rotor for 80 min at 4° C. The ribosomal pellet was suspended in 500 mm KCl in lysis buffer and centrifuged as described above. The resulting ribosomal salt wash enriched in eIF3 was incubated in batch for 1 h at 4° C with 0.8 ml of HIS-bindTM resin (Novagen) equilibrated with binding buffer (20 mm Tris-HCl, pH 7.9, 10% (v/v) glycerol, 30 mm imidazole, and 500 mm NaCl). After pouring the resin into a column, unbound proteins were removed by washing with 40 bed volumes of binding buffer, and eIF3 was eluted with the same buffer containing 500 mm imidazole. Eluted fractions were analyzed by SDS-PAGE and Western immunoblotting. Plasmid pRIB-S1 (kindly provided by J. Warner, Albert Einstein University) carrying a copy of the yeast rDNA gene was used as template to amplify two fragments of yeast 18 S rDNA overlapping at the unique SacI site. rDNA nucleotides 1–1248 were amplified with primers 5′-CCCCTCGAGTATCTGGTTGATCCTGCCAG-3′ (introducing anXhoI site (underlined) upstream of the first rDNA nucleotide) and 5′-CCCGAATTCGAGCTCTCAATCTGTCAATC-3′ (introducing an EcoRI site downstream of the SacI site in the rDNA). Nucleotides 1243–1800 were amplified with primers 5′-CCCTCGAGGAGCTCTTTCTTGATTTTGTG-3′ (introducing anXhoI site upstream of the SacI site) and 5′-CCCATCGATTAATGATCCTTCCGCAGGTT-3′ (introducing aClaI site after the last rRNA nucleotide). PCR products were cloned into pNoTA to generate pNo18S-5′ and pNo18S-3′, and the sequence was verified for both constructs. To construct templates for in vitro transcription under control of the T7 RNA polymerase promoter, the XhoI/EcoRI fragment from pNo18S-5′ was cloned into pSP73 digested with XhoI/EcoRI to generate pSP18S-5′. pSP18S-3′ was created by ligating theXhoI/ClaI fragment from pNo18S-3′ intoXhoI/ClaI-digested pSP73. Ligating aSacI/ClaI fragment from pNo18S-3′ into theSacI/ClaI sites of pSP18S-5′ resulted in pSP18S-fl. pSP18S-fl and pSP18S-3′ were linearized with ClaI and transcribed with T7 RNA polymerase to yield rRNA-(1–1800) and rRNA-(1243–1800), respectively. pSP18S-5′ digested withEcoRI and BstBI generated rRNA-(1–1248) and rRNA-(1–264), respectively. To synthesize rRNA-(263–1248), pSP18S-5′ was digested with XhoI and BstBI, blunt-ended with Klenow DNA polymerase, religated, and digested withEcoRI prior to transcription. All T7 transcripts carry the sequence 5′-GGGAGACCGGCCUCGAG at the 5′-end of the rRNAs, corresponding to the pSP73 sequence following the transcription start site. In vitro transcription was carried out in the presence of 50 μCi of [α-32P]UTP (800 Ci/mmol) with the T7/SP6 transcription MAXIscript kit (Ambion Inc.) according to the manufacturer's recommendations. Unincorporated nucleotides were removed using MicroSpinTM S-200 HR columns (Amersham Pharmacia Biotech). The 32P-labeled β-globin mRNA was prepared as described previously (25Wei C.-L. MacMillan S.E. Hershey J.W.B. J. Biol. Chem. 1995; 270: 5764-5771Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Isotopically labeled transcripts were analyzed on denaturing 4% (for long transcripts) or 5% (for shorter transcripts) polyacrylamide gels. For Northwestern RNA binding experiments, purified His6-p33 (3 μg), His6-p33ΔC (3 μg), and yeast lysate (10 μg) were subjected to SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. The membranes were treated for 20 min with binding buffer containing 20 mm HEPES-KOH, pH 7.5, 2 mm Mg(OAc)2, 75 mm KOAc, 1 mm EDTA, 1 mm dithiothreitol, 0.2% (w/v) CHAPS, 1 mg/ml E. coli tRNA, and 200 units of ribonuclease inhibitor (Amersham Pharmacia Biotech). Membranes were then incubated for 20 min with 200,000 cpm/ml of the 32P-labeled 18 S rRNA or β-globin transcripts in 8 ml of binding buffer. The blots were washed three times for 5 min each with binding buffer and subjected to autoradiography. When this work was initiated, the gene encoding the 33-kDa subunit of eIF3 had not been identified. An attempt to obtain partial amino acid sequences from tryptic digests of p33 from purified yeast eIF3 was not successful. However, at this time, partial amino acid sequence information was being developed in the laboratory (34Block K.L. Vornlocher H.-P. Hershey J.W.B. J. Biol. Chem. 1998; 273: 31901-31908Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) for one of the last subunits of mammalian eIF3 to be characterized, namely eIF3-p44. The mouse p44 amino acid sequence was used to search the yeast data base as described under “Materials and Methods,” and an ORF encoding a putative homolog of mammalian eIF3-p44 was identified. Since the yeast ORF appeared to encode a 30.5-kDa protein, we considered it to be a good candidate for the gene for yeast eIF3-p33, which we namedTIF35. Evidence reported below and elsewhere (18Phan L. Zhang X. Asano K. Anderson J. Vornlocher H.-P. Greenberg J.R. Goldfarb D.S. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 4935-4946Crossref PubMed Scopus (158) Google Scholar) shows that the protein product of TIF35 is a 33-kDa protein that is present in a complex with other known eIF3 subunits, confirming that the gene encodes the p33 subunit. A 2.1-kb fragment of DNA containing TIF35 was amplified by PCR from yeast genomic DNA and cloned into the pNoTA vector to yield pNo-TIF35 as described under “Materials and Methods.” The 2.1-kb DNA contains an ORF of 825 base pairs that codes for a protein of 274 amino acid residues with a calculated mass of 30,501 Da. The sequence context of the first AUG in the ORF, AUAAUG (the initiation codon is underlined), resembles the yeast consensus context, A(A/U)AAUG (26Cigan A.M. Donahue T.F. Gene (Amst.). 1987; 59: 1-18Crossref PubMed Scopus (257) Google Scholar). This AUG is preceded by an in-frame UAG termination codon, whereas the next in-frame AUG is found far downstream at codon 117. Thus, the first AUG very likely serves as the initiation codon. Hybridization of 32P-labeled DNA probes (derived from the coding region) to a single band of genomic DNA individually digested with four different restriction enzymes suggested the presence of a single gene locus (data not shown). Sequence comparisons revealed amino acid sequence identities/similarities of 35.8/46.9, 33.1/42.5, 33.3/43.1, and 33.6/47.3% when yeast eIF3-p33 was compared with the corresponding homologous proteins from Schizosaccharomyces pombe(GenBankTM/EBI accession number AB011823), human (U96074), mouse (AA109090, AA270800, and W18370), and Caenorhabditis elegans (Z50044; protein F22B5.2), respectively. When restricting the sequence comparison to the C-terminal 93-amino acid region of p33 that contains the RRM, identity/similarity values range between 43.7/56.3% for S. pombe and 36.4/47.7% for C. elegans. Therefore, eIF3-p33 appears to be present and moderately conserved in essentially all eukaryotic cells. In contrast, no homolog is found in Archaea sequences. To examine whether or notTIF35 is an essential gene, we constructed a diploid strain, PH33D-7, in which one of the TIF35 genes is nearly entirely deleted and is replaced by HIS3, as described under “Materials and Methods.” Tetrad analysis of PH33D-7 revealed that only two of the four spores in each of 30 asci formed colonies on rich medium (YPD), even after a long incubation at 30° C (data not shown). All viable spores were unable to grow on SD-His medium, suggesting that the phenotype oftif35::HIS3 is lethal. The segregation pattern of the tetrad spores (2+:2−) and the fact that no His+ segregants were found indicate thatTIF35 is necessary for germination and/or cell viability. To confirm that the lethal phenotype is due to disruption ofTIF35, plasmid pRS316-TIF35, which expressesTIF35 from its own promoter on a centromeric plasmid carrying a URA3 marker gene, was constructed and transformed into PH33D-7. Ura+ transformants were selected; two were sporulated; and the resulting asci were dissected. Two, three, or four viable spore colonies per ascus were obtained (data not shown). Only one spore from the three viable tetrads or two from the four viable tetrads grew on SD-His plates, and all His+ spore colonies were also Ura+. Thus, spores containing thetif35::HIS3 allele must harbor theURA3 plasmid, which carries TIF35. The results show that TIF35 is the only gene affected and that the disruption is complemented by the cloned gene. To obtain further evidence that TIF35 encodes a subunit of eIF3, we fused six histidine residues to the N terminus of p33 to create His6-p33. Strain PHL33, expressing His6-p33 as the sole source of this subunit, grows at the wild-type rate, indicating that the histidine tag is not deleterious to p33 function. Ribosomal salt wash fractions were prepared from strains expressing the wild-type (strain W303–1A) and histidine-tagged (strain PHL33) forms of p33, and the preparations were fract" @default.
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• W2066176289 title "Characterization of the p33 Subunit of Eukaryotic Translation Initiation Factor-3 from Saccharomyces cerevisiae" @default.
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