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 were 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 [rabbit and mouse] 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.