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 Mg-2+, ribosomes, and AUG codon on the Met-tRNAf Met-initiation factor-GTP complex were studied using a Millipore filtration method (J. Biol. Chem. 248, 4500 (1973)). Upon addition of increasing concentration of Mg-2+, the Met-tRNAf Met-initiation factor-GTP complex dissociates into free Met-tRNAf Met and initiation factor (GTP), with an infection around 1.5 to 2 mM Mg-2+. The Mg-2+-induced dissociation of Met-tRNAf Met-initiation factor-GTP complex was enhanced at ice bath temperature. At 37 degrees and in the presence of 1.5 to 2mM Mg-2+, the Met-tRNAf Met-initiation factor-GTP complex catalyzes the transfer of Met-tRNAf Met to ribosomes and AUG codon. Ribosome bound Met-tRNAf Met is stable to Mg-2+ and low temperature. A Millipore filtration assay for studies of (35S)Met-tRNAf Met binding to ribosomes and Aug codon has been developed. The assay procedure is carried out in three stages. In Stage I, the Met-tRNAf Met is bound to initiation factor in the presence of GTP, AUG codon (required for Stage II reaction), and 3.7 times 10-5 M aurintricarboxylic acid. The incubation is carried out at 37 degrees for 5 min. In Stage II, ribosomes and Mg-2+ (1.5 to 2mM final concentration) are added and the incubation is continued at 37 degrees for 10 min. In Stage III, more Mg-2+ is added to make the final Mg-2+ concentration of the incubation mixture 5 mM, and the reactions are further incubated at ice bath temperature for 10 min. The reactions are then terminated by addition of excess cold wash buffer and filtered through Millipore filters. 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 (35S)Met-tRNAf Met to Millipore filters was observed when both ribosomes and AUG codon were added. The Met-tRNAf 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-tRNAf Met binding activities separate into two forms, and are designated as IF-1A and IF-1B. These two forms can be distinguished by the stabilities of their respective Met-tRNAf Met-IF-1-GTP complexes to Mg-2+. The Met-tRNAf Met-IF-1A-GTP complex is distinctly more stable in the presence of Mg-2+ than Met-tRNAf Met-IF-1B-GTP complex. Continue.