Identification of initiator AUG binding proteins in initiation step of protein synthesis

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An 80S initiation complex was formed by incubation of a rabbit reticulocyte lysate with 5'terminal 70-nucleotide-long fragment of tabbaco mosaic virus RNA (TMV-RNA) without the cap and with its initiator AUG codon at 3'terminus, designated ad ^<32>P<OMEG>, which was labeled with ^<32>pCp. Labeled subfragments of ^<32>P<OMEGA>, which were protected from micrococcal nuclease digestion by 80S initiating ribosomes, were attached to 5SRNA-L5 protein particle (5SRNP). To determine initiator AUG binding proteins, initiation complexes (40S and 80S) were irradiated by UV light to induce crosslinks between ribosomal proteins and ^<32>p<OMEGA>. Analyses of the crosslinked ^<32>p-proteins showed that proteis S7, S10, S25, S29 and L5 in 40S initiation complex, and proteins S7, S25, S29 and L5 in 80S initiation complex. Analyses of a low molecular weight RNA in the 40S complex showed that 5SRNA was stably associated to the 40S subunits. The same result, association of 5SRNA to 40S subunits, was shown in native 40S subunits from liver of rat.