Protein synthesis in rabbit reticulocytes. Characteristics of mRNA (AUG codon) dependent binding of Met tRNA(f)(Met) to 40 S and 80 S ribosomes

The characteristics of Met tRNA(f)(Met) binding to ribosome (40 S and 80 S) were studied using a two stage assay method and the complexes formed were analyzed either by Millipore filtration or by sucrose density gradient centrifugation. The results are summarized as follows: (a) with both assay methods Met tRNA(f)(Met) binding to 40 S ribosomes was entirely dependent upon addition of a partially purified mixture of initiation factors and AUG codon; (b) this binding occurred over a wide Mg²⁺ concentration range; significant binding was observed even at 20 mM Mg²⁺; (c) upon addition of 60 S ribosomes, a significant part of Met tRNA(f)(Met) bound to 40 S ribosomes was transferred to 80 S complex. This transfer reaction had a sharp Mg²⁺ optimum around 2 mM. Met tRNA(f)(Met).80 S .AUG complex thus formed was active in Met puromycin synthesis; (d) Met tRNA(f)(Met) deacylase present in crude 0.5 M KCl ribosomal wash is a potent inhibitor of the binding reaction as it deacylates Met tRNA(f)(Met) in the Met tRNA(f)(Met).40 S .AUG complex; (e) glutaraldehyde (0.5%) degrades Met tRNA(f)(Met).40 S .AUG complex but increases the background binding of Met tRNA(f)(Met) to 40 S ribosomes in the absence of AUG codon; (f) polynucleotides containing uracil and adenosine are strong inhibitors of Met tRNA(f)(Met) binding to 40 S ribosomes. The order of inhibitory activities of the polynucleotides tested was as follows: poly(rU) poly(rA) (2:1) > poly(rU).poly(rA) (1:1 > poly(rU) > poly(rA). Other RNAs tested such as poly(rC), poly(rI).poly(rC) and phi6 bacteriophage RNA (double stranded) were without significant effects on the Met tRNA(f)(Met) binding reaction.