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In vitro suppression as a tool for the investigation of translation initiation
V A Karginov1, S V Mamaev, S M Hecht
1Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22901, USA.
Abstract:
An in vitro protein synthesizing system that employs rabbit reticulocyte lysates has been employed for protein production from mRNAs containing nonsense (UAG) codons in the presence of misacylated suppressor tRNAs.The system includes a misacylated Escherichia coli tRNAAlaCUA that functions at least as efficiently as any suppressor tRNA transcript reported to date and which has been shown not to be a substrate for (re)activation by alanyl-tRNA synthetase. Application of the optimized system for preparation of dihydrofolate analogs has also permitted analysis of competing mechanisms that control the sites(s) of translation initiation.