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Journal of Biochemistry 2005 138(1):41-46; doi:10.1093/jb/mvi096
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© 2005 The Japanese Biochemical Society

Regular Paper

Ribosomal Proteins Cross-Linked to the Initiator AUG Codon of a mRNA in the Translation Initiation Complex by UV-Irradiation

Yoshiaki Takahashi1,*, Sigeru Hirayama2 and Shoji Odani3

1 Department of Medical Technology, School of Health Sciences, Faculty of Medicine, Niigata University, Asahimachi-Dori 2-746, Niigata 951-8518; 2 Asahimachi Laboratory of Radioisotope Research, Asahimachi-Dori 1-757; and 3 Faculty of Science, Niigata University, Ikarashi 2-8050, Niigata 950-2181

* To whom correspondence should be addressed. Tel/Fax: +81-25-227-0959, E-mail: yoshiaki{at}clg.niigata-u.ac.jp

Eukaryotic ribosomal proteins constituting the binding site for the initiator codon AUG on the ribosome at the translation initiation step were investigated by UV-induced cross-linking between protein and mRNA. The 80S-initiation complex was formed in a rabbit reticulocyte cell–free system in the presence of sparsomycin with radiolabeled {Omega}-fragment as a template, which was a 73-base 5'-leader sequence of tobacco mosaic virus RNA having AUG at the extreme 3'-terminal end and extended with 32pCp. Two radioactive peaks were sedimented by sucrose gradient centrifugation, one being the 80S initiation complex formed at the 3'-terminal AUG codon, and the other presumably a "disome" with an additional 80S ribosome bound at an upstream AUU codon, formed when {Omega}-fragment was incubated with sparsomycin [Filipowicz and Henni (1979) Proc. Natl. Acad. Sci. USA 76, 3111–3115]. Cross-links between ribosomal proteins and the radiolabeled {Omega}-fragment were induced in situ by UV-irradiation at 254 nm. After extensive nuclease digestion of the complexes, ribosomal proteins were separated by two-dimensional gel electrophoresis. Autoradiography identified the proteins S7, S10, S25, S29, and L5 of the 80S initiation complex and S7, S25, S29 and L5 of that in the disome as 32P-labeled proteins. Together with the results of cross-linking experiments of other investigators and recently solved crystal structures of prokaryotic ribosomes, the spatial arrangement of eukaryotic ribosomal proteins at the AUG-binding domain is discussed.


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