Protein synthesis in rabbit reticulocytes. A study of Met tRNA(f)(met) binding factor(s) and Met tRNA(f)(met) binding to ribosomes and AUG codon

The effects of additions of Mg²⁺, ribosomes, and AUG codon on the Met,tRNA(f,Met) initiation factor GTP complex were studied using a Millipore filtration method. Upon addition of increasing concentration of Mg²⁺, the Met,tRNA(f,Met) initiation factor GTP complex dissociates into free Met,tRNA(f,Met) and initiation factor (GTP), with an inflection around 1.5 to 2 mg Mg²⁺. The Mg²⁺ induced dissociation of Met,tRNA(f,Met) initiation factor GTP complex was enhanced at ice bath temperature. At 37° and in the presence of 1.5 to 2 mM Mg²⁺, the Met,tRNA(f,Met) initiation factor GTP complex catalyzes the transfer of Met,tRNA(f,Met) to ribosomes and AUG codon. Ribosome bound Met,tRNA(f,Met) is stable to Mg²⁺ and low temperature. A Millipore filtration assay for studies of [³⁵]Met,tRNA(f,Met) binding to ribosomes and AUG codon has been developed. Under the standard assay conditions, the radioactivity bound to Millipore filters in the absence of ribosomes and AUG codon is markedly reduced. Addition of ribosomes alone gave a significant increase in the radioactivity bound to Millipore filters. A further 2 to 3 fold stimulation of binding of [³⁵]Met,tRNA(f,Met) to Millipore filters was observed when both ribosomes and AUG codon were added. The Met,tRNA(f,Met) bound to ribosomes under the assay condition was reactive with puromycin. Upon DEAE cellulose chromatography of a partially purified mixture of initiation factors (IF), Met,tRNA(f,Met) binding activities separate into 2 forms, and are designated as IF,1A and IF,1B. These 2 forms can be distinguished by the stabilities of their respective Met,tRNA(f,Met) IF,1 GTP complexes to Mg²⁺. The Met,tRNA(f,Met) IF,1A GTP complex is distinctly more stable in the presence of Mg²⁺ than Met,tRNA(f,Met) IF,1B GTP complex. Sucrose density gradient analysis of these complexes revealed that the approximate molecular weight of the Met,tRNA(f,Met) IF,1A GTP complex is 160,000 and that of Met,tRNA(f,Met) IF,1B GTP complex is 65,000. Both IF,1A and IF,1B catalyze the binding of Met,tRNA(f,Met) to ribosomes and addition of AUG codon does not have any significant effect on such binding. The maximum stimulation of AUG directed Met,tRNA(f,Met) binding to ribosomes was observed when all 3 factors, IF,1A (or IF,1B), IF 2, and IF 3 are added together.