Preparation and activity of synthetic unmodified mammalian tRNA(i)(Met) ininitiation of translation in vitro

Translation of eukaryotic mRNA is initiated by a, unique amino-acyl tRNA, M et-tRNA(i)(Met), Which passes through a complex series of highly specific i nteractions with components of the translation apparatus during the initiat ion process. To facilitate in vitro biochemical and molecular biological an alysis of these interactions in fully reconstituted translation initiation reactions, we generated mammalian tRNA(i)(Met) by in vitro transcription th at lacked all eight base modifications present in native tRNA(i)(Met). Here we report a method for in vitro transcription and aminoacylation of synthe tic unmodified initiator tRNA(i)(Met) that is active in every stage of the initiation process, including aminoacylation by methionyl-tRNA synthetase, binding of Met-tRNA(i)(Met) to eIF2-GTP to form a ternary complex, binding of the ternary complexes to 40S ribosomal subunits to form 43S complexes, b inding of the 43S complex to a native capped eukaryotic mRNA, and scanning on its 5' untranslated region to the correct initiation codon to form a 48S complex, and finally joining with a 60S subunit to assemble an 80S ribosom e that is competent to catalyze formation of the first peptide bond using t he [S-35] methionine residue attached to the acceptor terminus of the tRNA, Met transcript.