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Unformatted text preview: letters to nature NATURE | VOL 403 | 20 JANUARY 2000 | www.nature.com 335 Western blotting Ribosomal complexes assembled and purified as described above were TCA-precipitated. Proteins were resolved on 12% polyacrylamide gel, transferred to nitrocellulose mem- brane and probed for eIF1 and eIF5B using T7-tag antibodies (Novagen) and for eIF2 a and eIF3 (p170) using specific antibodies. Received 13 September; accepted 16 November 1999. 1. Merrick, W. C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56, 291– 315 (1992). 2. Pestova, T. V., Borukhov, S. I. & Hellen, C. U. T. Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons. Nature 394, 854–859 (1998). 3. Chakrabarti, A. & Maitra, U. Functions of eukaryotic initiation factor 5 in the formation of an 80S ribosomal polypeptide chain initiation complex. J. Biol. Chem. 266, 14039–14045 (1991). 4. Das, K., Chesevich, J. & Maitra, U. Molecular cloning and expression of cDNA for mammalian translation initiation factor 5. Proc. Natl Acad. Sci. USA 90, 3058–3062 (1993). 5. Huang, H.-K., Yoon, H., Hannig, E. M. & Donahue, T. F. GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae . Genes Dev. 11, 2396– 2413 (1997). 6. Choi, S. K., Lee, J. H., Zoll, W. L., Merrick, W. C. & Dever, T. E. Promotion of Met-tRNA Met binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast. Science 280, 1757–1760 (1998). 7. Lee, J. H., Choi, S. K., Roll-Mecak, A., Burley, S. K. & Dever, T. E. Universal conservation in translation initiation revealed by human and archaeal homologs of bacterial translation factor IF2. Proc. Natl Acad. Sci. USA 96, 4342–4347 (1999). 8. Sacerdot, C., Dessen, P., Hershey, J. W. B., Plumbridge, J. A. & Grunberg-Manago, M. Sequence of the initiation factor IF2 gene; unusual protein features and homologies with elongation factors. Proc. Natl Acad. Sci. USA 81, 7787–7791 (1984). 9. Kolakofsky, D., Dewey, K. F., Hershey, J. W. B. & Thach, R. E. Guanosine 5 9-triphosphatase activity of initiation factor f2. Proc. Natl Acad. Sci. USA 61, 1066–1070 (1968). 10. Godefroy-Colburn, T. et al . Light-scattering studies showing the effect of initiation factors on the reversible dissociation of Escherichia coli ribosomes. J. Mol. Biol. 94, 461–478 (1975). 11. Luchin, S. et al . In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2. Direct evidence that GTP hydrolysis is necessary for factor recycling. J. Biol. Chem. 274, 6074–6079 (1999). 12. Lockwood, A. H., Sarkar, P. & Maitra, U. Release of polypeptide chain initiation factor IF-2 during initiation complex formation. Proc. Natl Acad. Sci. USA 69, 3602–3605 (1972)....
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