The role of complexes between the 40-S ribosomal subunit and Met-tRNA-Met-f in the initiation of protein synthesis in the wheat-germ system.

We have demonstrated the formation of a complex between the 40-S ribosomal subunit and Met-tRNAMetf in the absence of mRNA in an unfractionated wheat-germ system. Two types of experiment indicate that these complexes (40S · Met-tRNAMetf) are true intermediates in the formation of 80-S initiation complexes containing mRNA, Met-tRNAMetf and both ribosomal subunits. Firstly, isolated 40S · Met-tRNAMetf complexes were used preferentially over free Met-tRNAMetf in the formation of 80-S initiation complexes on addition of mRNA. Secondly by the use of methioninol-AMP, an inhibitor of methionyl-tRNA synthetase, to deplete endogenous Met-tRNAMetf pools, we showed that the binding of mRNA to ribosomes was strictly dependent on the presence of Met-tRNAMetf. The 40S · Met-tRNAMetf complexes had other properties consistent with their role as intermediates in initiation. The radioactivity in 40S · Met-tRNAMetf complexes labelled with [35S]Met-tRNAMetf was shifted into 80-S initiation complexes on addition of mRNA. This shift was prevented by aurintricarboxylic acid, which inhibits the binding of mRNA to ribosomes. In the presence of edeine, which blocks the binding of the 60-S subunit, the radioactivity was found in complexes containing mRNA, Met-tRNAMetf and 40-S subunits. These results were confirmed by the use of labelled mRNA. We propose that the mechanism of protein chain initiation is universal to all cells, the first step being the formation of a complex between the small ribosomal subunit and (f)Met-tRNAMetf. This complex can then bind mRNA in a process which must to some extent involve selection of the initiator AUG codon through interactions with the anticodon of the (f)Met-tRNAMetf. The binding of mRNA is followed by the addition of the large ribosomal subunit and the formation of an initiation complex capable of forming the first peptide bond.

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