Preparation and activity of synthetic unmodified mammalian tRNAi Met in initiation of translation in vitro

Abstract

Translation of eukaryotic mRNA is initiated by a unique amino-acyl tRNA, Met-tRNA i Met , which passes through a complex series of highly specific interactions with components of the translation apparatus during the initiation process. To facilitate in vitro biochemical and molecular biological analysis of these interactions in fully reconstituted translation initiation reactions, we generated mammalian tRNA iMet by in vitro transcription that lacked all eight base modifications present in native tRNA i Met . Here we report a method for in vitro transcription and aminoacylation of synthetic unmodified initiator tRNA iMet 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, binding of the 43S complex to a native capped eukaryotic mRNA, and scanning on its 59 untranslated region to the correct initiation codon to form a 48S complex, and finally joining with a 60S subunit to assemble an 80S ribosome that is competent to catalyze formation of the first peptide bond using the [ 35 S]methionine residue attached to the acceptor terminus of the tRNA i Met transcript.

One Single In-frame AUG Codon Is Responsible for a Diversity of Subcellular Localizations of Glutaredoxin 2 in Saccharomyces cerevisiae

“…However, if the AUG is located in a suboptimal nucleotide sequence context, it may be missed and the ribosome would slide downstream to initiate translation at the next AUG. Another model for initiation of translation postulates direct entry of the ribosome at internal AUG triplets (internal ribosomal entry site) (45). In GRX2, the second TSS is present at an in-frame AUG, representing the amino acid residue Met 35 .…”

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

“…For this work we haveused tRNAs transcribed in vitro,w hich allowed us to generate and study al arge number of variant initiator ande longator tRNAs, but didn ot allow us to probe the roles of basem odifications in them. However, previous work has failed to find any detectable differences between the unmodifieda nd native initiator tRNA in av ariety of in vitro assays ( Pestova and Hellen 2001;Kapp and Lorsch 2004a).…”

tRNA modifications: Necessary for correct tRNA‐derived fragments during the recovery from stress?

“…Since even scarcely modified (mitochondrial) or even unmodified (e.g. in vitro transcribed) tRNAs can contribute to protein synthesis [11], it follows that many tRNA modifications are not essential for tRNA function during protein synthesis. If so, why are tRNAs so heavily modified?…”