title: Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes.
abstract: Proteins that are located adjacent to the 5' end of mRNA in initiation complexes have been detected by chemical crosslinking. Reovirus mRNA containing radioactivity exclusively in the [3H]methyl-labeled "cap," m7G(5')ppp(5')-Gm, was oxidized with sodium periodate to convert the 2',3'-cis-diol of the 5'-terminal m7G to a reactive dialdehyde. Oxidized mRNA was incubated in cell-free protein-synthesizing systems derived from wheat germ or mammalian cells, and the resulting mRNA-ribosome initiation complexes were reduced with NaBH3CN. By this chemical procedure, putative Schiff bases between mRNA 5'termini and amino groups of neighboring proteins were stabilized by reduction, yielding covalently linked protein-RNA conjugates. Under conditions of ribosome binding, a limited number of polypeptides associated with the mRNA-ribosome complexes were crosslinked, suggesting that these proteins are positioned near and may interact with the 5' end of mRNA during initiation. This method should also be useful for studying the spatial relationships between molecules in other similar nucleoprotein complexes.
mesh_terms: D000818:Animals; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010936:Plant Extracts / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D011817:Rabbits; D012087:Reoviridae / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
chemical_list: D010448:Peptide Initiation Factors; D010455:Peptides; D010936:Plant Extracts; D012333:RNA, Messenger; D012367:RNA, Viral
references.citation: Virology. 1977 Apr;77(2):566-78
references.pmid: 855185
references.citation: Nature. 1977 Mar 17;266(5599):235-9
references.pmid: 557727
references.citation: Proc Natl Acad Sci U S A. 1976 Oct;73(10):3448-52
references.pmid: 185613
references.citation: Nucleic Acids Res. 1977 Sep;4(9):3065-81
references.pmid: 909799
references.citation: Proc Natl Acad Sci U S A. 1976 Sep;73(9):3005-9
references.pmid: 1067598
references.citation: Cell. 1976 Dec;9(4 PT 2):645-53
references.pmid: 1017010
references.citation: Biochemistry. 1976 Dec 28;15(26):5761-8
references.pmid: 1009086
references.citation: J Mol Biol. 1973 Feb 19;73(3):329-49
references.pmid: 4686199
references.citation: Methods Enzymol. 1974;30:101-27
references.pmid: 4852488
references.citation: J Virol. 1972 Apr;9(4):636-45
references.pmid: 4336562
references.citation: J Mol Biol. 1975 Apr 15;93(3):391-404
references.pmid: 49436
references.citation: J Mol Biol. 1975 Dec 15;99(3):401-18
references.pmid: 1214295
references.citation: Proc Natl Acad Sci U S A. 1976 Aug;73(8):2584-8
references.pmid: 1066667
references.citation: FEBS Lett. 1976 May 1;64(2):326-31
references.pmid: 179883
references.citation: Biochem Biophys Res Commun. 1974 Feb 4;56(3):745-52
references.pmid: 4363751
references.citation: J Mol Biol. 1976 Jul 5;104(3):637-58
references.pmid: 950671
references.citation: Cell. 1975 Oct;6(2):185-95
references.pmid: 1182800
references.citation: Nature. 1975 May 1;255(5503):33-7
references.pmid: 165427
references.citation: Proc Natl Acad Sci U S A. 1975 Mar;72(3):1189-93
references.pmid: 165487
references.citation: J Mol Biol. 1973 Nov 15;80(4):575-99
references.pmid: 4204102
references.citation: Nature. 1976 Jul 1;262(5563):32-7
references.pmid: 180425
references.citation: Eur J Biochem. 1974 Jul 1;46(1):83-8
references.pmid: 4850204
references.citation: Biochem Biophys Res Commun. 1973 Apr 16;51(4):932-8
references.pmid: 4703562
references.citation: Proc Natl Acad Sci U S A. 1976 May;73(5):1559-63
references.pmid: 1064023
references.citation: FEBS Lett. 1976 Feb 15;62(2):111-4
references.pmid: 767141
references.citation: Proc Natl Acad Sci U S A. 1976 Jan;73(1):19-23
references.pmid: 1061116
references.citation: J Biol Chem. 1976 Jul 25;251(14):4259-66
references.pmid: 932032
references.citation: Nature. 1976 Mar 25;260(5549):359-60
references.pmid: 1256580
references.citation: Nature. 1976 May 27;261(5558):291-4
references.pmid: 944862
references.citation: FEBS Lett. 1975 Oct 15;58(1):281-4
references.pmid: 1225593
references.citation: J Biol Chem. 1970 Mar 10;245(5):955-61
references.pmid: 5417266