NDLI: Preparation and activity of synthetic unmodified mammalian tRNAi(Met) in initiation of translation in vitro.

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

Content Provider	Semantic Scholar
Copyright Year	2001
Abstract	Translation of eukaryotic mRNA is initiated by a unique amino-acyl tRNA, Met-tRNAi(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 tRNAi(Met) by in vitro transcription that lacked all eight base modifications present in native tRNAi(Met). Here we report a method for in vitro transcription and aminoacylation of synthetic unmodified initiator tRNAi(Met) that is active in every stage of the initiation process, including aminoacylation by methionyl-tRNA synthetase, binding of Met-tRNAi(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 5' 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 [35S]methionine residue attached to the acceptor terminus of the tRNAi(Met) transcript.
File Format	PDF HTM / HTML
Alternate Webpage(s)	http://rnajournal.cshlp.org/content/7/10/1496.full.pdf
PubMed reference number	11680854v1
Volume Number	7
Issue Number	10
Journal	RNA
Language	English
Access Restriction	Open
Subject Keyword	5' Untranslated Regions Amino Acyl-tRNA Synthetases Aminoacylation Codon, Initiator Genetic Translation Process In Vitro [Publication Type] Mammals Methionine-tRNA Ligase Ribosome Subunits Ribosome Subunits, Small Ribosomes Transcript Transcription Initiation Translation Initiation
Content Type	Text
Resource Type	Article

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