Promotion of Met-tRNA(i)/(Met) binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast

Science

Volumn 280, Issue 5370, 1998, Pages 1757-1760

  Choi, Sang Ki ^a   Lee, Joon H ^a   Zoll, Wendy L ^b   Merrick, William C ^b   Dever, Thomas E ^a  

^a EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT   (United States)

^b CASE WESTERN RESERVE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

GLUTATHIONE TRANSFERASE; HYBRID PROTEIN; INITIATION FACTOR 2; MESSENGER RNA; METHIONINE TRANSFER RNA; PROTEIN; DNA BINDING PROTEIN; FUNGAL PROTEIN; INITIATION FACTOR; MET TRNA(I)(MET); MET-TRNA(I)(MET); PROTEIN KINASE; SACCHAROMYCES CEREVISIAE PROTEIN;

ARTICLE; GROWTH; NONHUMAN; OPEN READING FRAME; PHENOTYPE; POLYSOME; PRIORITY JOURNAL; PROTEIN SYNTHESIS; RIBOSOME; RIBOSOME SUBUNIT; RNA BINDING; SACCHAROMYCES CEREVISIAE; TRANSLATION INITIATION; BIOSYNTHESIS; CHEMISTRY; CYTOPLASM; GENETICS; METABOLISM; RNA TRANSLATION; START CODON;

CODON, INITIATOR; CYTOPLASM; DNA-BINDING PROTEINS; EUKARYOTIC INITIATION FACTOR-2; FUNGAL PROTEINS; PEPTIDE CHAIN INITIATION, TRANSLATIONAL; PEPTIDE INITIATION FACTORS; PROKARYOTIC INITIATION FACTOR-2; PROTEIN BIOSYNTHESIS; PROTEIN KINASES; RECOMBINANT FUSION PROTEINS; RIBOSOMES; RNA, TRANSFER, MET; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS;

EID: 0032510912     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5370.1757     Document Type: Article

References (38)

1. U. Maitra, E. A. Stringer, A. Chaudhuri, Annu. Rev. Biochem. 51, 869 (1982); J. W. B. Hershey, in Translation in Eukaryotes, H. Trachsel, Ed. (CRC Press, Boca Raton, FL, 1991) pp. 353-374.
2. U. Maitra, E. A. Stringer, A. Chaudhuri, Annu. Rev. Biochem. 51, 869 (1982); J. W. B. Hershey, in Translation in Eukaryotes, H. Trachsel, Ed. (CRC Press, Boca Raton, FL, 1991) pp. 353-374.
3. W. C. Merrick, Microbiol. Rev. 56, 291 (1992).
4. A. M. Cigan, L. Feng, T. F. Donahue, Science 242, 93 (1988).
5. T. F. Donahue, A. M. Cigan, E. K. Pabich, B. Castilho-Valavicius, Cell 54, 621 (1988); A. M. Cigan, E. K. Pabich, L. Feng, T. F. Donahue, Proc. Natl. Acad. Sci. U.S.A. 86, 2784 (1989); H. J. Yoon and T. F. Donahue, Mol. Cell. Biol. 12, 248 (1992); H.-k. Huang, H. Yoon, E. M. Hannig, T. F. Donahue, Genes Dev. 11, 2396 (1997).
6. T. F. Donahue, A. M. Cigan, E. K. Pabich, B. Castilho-Valavicius, Cell 54, 621 (1988); A. M. Cigan, E. K. Pabich, L. Feng, T. F. Donahue, Proc. Natl. Acad. Sci. U.S.A. 86, 2784 (1989); H. J. Yoon and T. F. Donahue, Mol. Cell. Biol. 12, 248 (1992); H.-k. Huang, H. Yoon, E. M. Hannig, T. F. Donahue, Genes Dev. 11, 2396 (1997).
7. T. F. Donahue, A. M. Cigan, E. K. Pabich, B. Castilho-Valavicius, Cell 54, 621 (1988); A. M. Cigan, E. K. Pabich, L. Feng, T. F. Donahue, Proc. Natl. Acad. Sci. U.S.A. 86, 2784 (1989); H. J. Yoon and T. F. Donahue, Mol. Cell. Biol. 12, 248 (1992); H.-k. Huang, H. Yoon, E. M. Hannig, T. F. Donahue, Genes Dev. 11, 2396 (1997).
8. T. F. Donahue, A. M. Cigan, E. K. Pabich, B. Castilho-Valavicius, Cell 54, 621 (1988); A. M. Cigan, E. K. Pabich, L. Feng, T. F. Donahue, Proc. Natl. Acad. Sci. U.S.A. 86, 2784 (1989); H. J. Yoon and T. F. Donahue, Mol. Cell. Biol. 12, 248 (1992); H.-k. Huang, H. Yoon, E. M. Hannig, T. F. Donahue, Genes Dev. 11, 2396 (1997).
9. H. Bussey et al., Proc. Natl. Acad. Sci. U.S.A. 92, 3809 (1995).
10. B. E. Diehl and J. R. Pringle, Genetics 127, 287 (1991).
11. C. J. Bult et al., Science 273, 1058 (1996); P. P. Dennis, Cell 89, 1007 (1997).
12. C. J. Bult et al., Science 273, 1058 (1996); P. P. Dennis, Cell 89, 1007 (1997).
13. S. Laalami, H. Putzer, J. A. Plumbridge, M. Grunberg-Manago, J. Mol. Biol. 220, 335 (1991).
14. S. K. Choi, J. H. Lee, T. E. Dever, data not shown.
15. 2 spores showing a marked slow-growth phenotype. Disruption of FUN12 in the slow-growing spores was confirmed by polymerase chain reaction (PCR) and could be complemented by transforming with a low-copy number plasmid carrying wild-type FUN12. Two fun12::LEU2 ascospores from the same tetrad were isolated and designated J133 and J135.
16. A. Vambutas, S. H. Ackerman, A. Tzagoloff, Eur. J. Biochem. 201, 643 (1991).
17. 2,1 mM dithiothreitol (DTT), 5 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, 5 μM leupeptin, 0.15 μM aprotinin; then they were centrifuged in an SW41 rotor at 4°C at 39,000 rpm for 2 hours 50 min. After ultracentrifugation, the gradients were unloaded with an ISCO VA-5 gradient collector and 0.6-ml fractions were collected while optical density was monitored at 254 nm.
18. T. E. Martin and L. H. Hartwell, J. Biol. Chem. 245, 1504 (1970); T. E. Martin, Exp. Cell Res. 80, 496 (1973).
19. T. E. Martin and L. H. Hartwell, J. Biol. Chem. 245, 1504 (1970); T. E. Martin, Exp. Cell Res. 80, 496 (1973).
20. 2. Capped and polyadenylated luciferase mRNA was prepared with the T7 luciferase (T7LUC) vector pRG166 (a gift of S. Green and Charles Moehle, RiboGene, Hayward, CA) and the Ampliscribe T7 transcription kit (Epicentre Technologies). mRNA was purified with the RNeasy total RNA kit (Qiagen). Translation assays were performed as described (27), except that the micrococcal nuclease treatment of the translation extracts was omitted. Luminescence was measured by adding 10 μl of translation mix to 100 μl of LUC assay reagent (Promega) and measuring the emission for 10 s on a Turner TD-20e luminometer.
21. To express the GST-ylF2 fusion protein in yeast under control of the yeast GAL1 promoter, we inserted a portion of the FUN12 cDNA from the plasmid fun12-1 [P. Sutrave, B. K. Shafer, J. N. Strathern, S. H. Hughes, Gene 146, 209 (1994); a gift from P. Sutrave and S. Hughes, National Cancer Institute, Frederick, MD] into the yeast GST fusion expression vector pEGKT [ D. A. Mitchell, T. K. Marshall, R. J. Deschenes, Yeast 9, 715 (1993)], creating the plasmid pC485. GST and GST-ylF2 were purified from the fun12Δ strain J130 (22) transformed with the plasmids pEGKT and pC485, respectively. The GST-ylF2 fusion protein containing ylF2 amino acid residues 396 to 1002, starting 16 residues before the GTP binding domain and extending to the COOH-terminus of the protein, was able to fully complement the slow-growth phenotype of the fun12Δ strain, indicating that the fusion protein is functional in vivo. Crude protein extracts from transformants grown in synthetic minimal medium containing 10% galactose and 2% raffinose were incubated with glutathione Sepharose 4B and washed; the GST and GST-ylF2 proteins were eluted in buffer containing 10 mM reduced glutathione. The purified proteins were dialyzed against sample buffer [20 mM tris-HCl (pH 7.5), 100 mM KCl, 1 mM DTT, 10% (v/v) glycerol] and stored in liquid nitrogen. Purified yeast elF2 [ G. D. Pavitt, K. V. A. Ramaiah, S. R. Kimball, A. G. Hinnebusch, Genes Dev. 12, 514 (1998)] was kindly provided by Graham Pavitt.
22. To express the GST-ylF2 fusion protein in yeast under control of the yeast GAL1 promoter, we inserted a portion of the FUN12 cDNA from the plasmid fun12-1 [P. Sutrave, B. K. Shafer, J. N. Strathern, S. H. Hughes, Gene 146, 209 (1994); a gift from P. Sutrave and S. Hughes, National Cancer Institute, Frederick, MD] into the yeast GST fusion expression vector pEGKT [ D. A. Mitchell, T. K. Marshall, R. J. Deschenes, Yeast 9, 715 (1993)], creating the plasmid pC485. GST and GST-ylF2 were purified from the fun12Δ strain J130 (22) transformed with the plasmids pEGKT and pC485, respectively. The GST-ylF2 fusion protein containing ylF2 amino acid residues 396 to 1002, starting 16 residues before the GTP binding domain and extending to the COOH-terminus of the protein, was able to fully complement the slow-growth phenotype of the fun12Δ strain, indicating that the fusion protein is functional in vivo. Crude protein extracts from transformants grown in synthetic minimal medium containing 10% galactose and 2% raffinose were incubated with glutathione Sepharose 4B and washed; the GST and GST-ylF2 proteins were eluted in buffer containing 10 mM reduced glutathione. The purified proteins were dialyzed against sample buffer [20 mM tris-HCl (pH 7.5), 100 mM KCl, 1 mM DTT, 10% (v/v) glycerol] and stored in liquid nitrogen. Purified yeast elF2 [ G. D. Pavitt, K. V. A. Ramaiah, S. R. Kimball, A. G. Hinnebusch, Genes Dev. 12, 514 (1998)] was kindly provided by Graham Pavitt.
23. To express the GST-ylF2 fusion protein in yeast under control of the yeast GAL1 promoter, we inserted a portion of the FUN12 cDNA from the plasmid fun12-1 [P. Sutrave, B. K. Shafer, J. N. Strathern, S. H. Hughes, Gene 146, 209 (1994); a gift from P. Sutrave and S. Hughes, National Cancer Institute, Frederick, MD] into the yeast GST fusion expression vector pEGKT [ D. A. Mitchell, T. K. Marshall, R. J. Deschenes, Yeast 9, 715 (1993)], creating the plasmid pC485. GST and GST-ylF2 were purified from the fun12Δ strain J130 (22) transformed with the plasmids pEGKT and pC485, respectively. The GST-ylF2 fusion protein containing ylF2 amino acid residues 396 to 1002, starting 16 residues before the GTP binding domain and extending to the COOH-terminus of the protein, was able to fully complement the slow-growth phenotype of the fun12Δ strain, indicating that the fusion protein is functional in vivo. Crude protein extracts from transformants grown in synthetic minimal medium containing 10% galactose and 2% raffinose were incubated with glutathione Sepharose 4B and washed; the GST and GST-ylF2 proteins were eluted in buffer containing 10 mM reduced glutathione. The purified proteins were dialyzed against sample buffer [20 mM tris-HCl (pH 7.5), 100 mM KCl, 1 mM DTT, 10% (v/v) glycerol] and stored in liquid nitrogen. Purified yeast elF2 [ G. D. Pavitt, K. V. A. Ramaiah, S. R. Kimball, A. G. Hinnebusch, Genes Dev. 12, 514 (1998)] was kindly provided by Graham Pavitt.
24. W. C. Merrick, Methods Enzymol. 60, 108 (1979); S. L. Adams, B. Safer, W, F. Anderson, W. C. Merrick, J. Biol. Chem. 250, 9083 (1975).
25. W. C. Merrick, Methods Enzymol. 60, 108 (1979); S. L. Adams, B. Safer, W, F. Anderson, W. C. Merrick, J. Biol. Chem. 250, 9083 (1975).
26. W. L. Zoll, S. K. Choi, T. E. Dever, W. C. Merrick, data not shown.
27. A. G. Hinnebusch, J. Biol. Chem. 272, 21661 (1997).
28. N. C. Kyrpides and C. R. Woese, Proc. Natl. Acad. Sci. U.S.A. 95, 224 (1998).
29. W. C. Merrick and W. F. Anderson, J. Biol. Chem. 250, 1107 (1975).
30. S. Z. Tarun and A. B. Sachs, Genes Dev. 9, 2997 (1995).
31. The plasmid pC482 was generated by inserting 1.3 kb of FUN12 5′ flanking and 0.9 kb of FUN12 3′ flanking DNA into the hisG::URA3::hisG plasmid pNKY51 [E. Alani, L. Cao, N. Kleckner, Genetics 116, 541 (1987)]. The fun12Δ strain J130 (leu2-3 leu2-112 ura3-52 fun12::hisG) is an ascospore product from the diploid strain H1056 that had been transformed with an Sph I-Mun I fragment from pC482.
32. R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989).
33. We inserted a 3.55-kb Sal I-Bam HI fragment from pC473 (10) into the corresponding sites of the low-copy-number URA3 vector pRS316 (23) generating the plasmid pC476. We extended the FUN12 insert in pC476 an additional 400 base pairs at the 5′ end by inserting a 1.56-kb PCR-generated Sal I-Eco 47III DNA fragment, creating the plasmid pC479. A Sal I-Bam HI fragment from pC479 containing the fulllength FUN12 DNA sequences was inserted between the same sites of the low-copy-number LEU2 vector pRS315 (23), creating the plasmid pC480.
34. - segregant, generating the haploid strain J129 (Matα leu2-3 leu2-112 ura3-52 fun12::leu2::hisG).
35. S. U. Astrom, U. von Pawel-Rammingen, A. S. Bystrom, J. Mol. Biol. 233, 43 (1993).
36. T. E. Dever, W. Yang, S. Astrom, A. S. Bystrom, A. G. Hinnebusch, Mol. Cell Biol. 15, 6351 (1995).
37. C. M. Moehle and A. G. Hinnebusch, ibid., 11, 2723 (1991).
38. We thank G. Pavitt for purified yeast elF2, M. Cigan for the IMT4 plasmid, C. Moehle and S. Green for plasmid pRG166, P. Sutrave and S. Hughes for the FUN12 cDNA clone, and A. Sachs for advice on the yeast in vitro translation assays; A. Hinnebusch, G. Pavitt, J. Anderson, and L. Phan and members of the Dever and Hinnebusch laboratories for helpful discussions; and A. Hinnebusch and R. Rolfes for comments on the manuscript. We thank T. Donahue for discussions and for communicating results before publication. Supported in part by National Institutes of Health grant 26796 (W.C.M.) and a training grant in cellular and molecular biology (GM08056 to W.L.Z.).