Abstract
This chapter describes the affinity chromatography for one-step purification of mammalian tRNA isoacceptors. Thermostable elongation factor Tu is immobilized on sepharose 48 using the cyanogen bromide coupling procedure. Of 20 mg Tu-GDP attached to 1 ml sepharose, about one percent was active in binding aminoacyl-tRNA. Immobilized bacterial elongation factor Tu that selectively binds aminoacyl-tRNAs is used as an affinity matrix. To increase the stability of the affinity columns, the elongation factor Tu is isolated from the extreme thermophilic eubacterium Thermus thermophilus . Isolation of a single group of tRNA isoacceptors by affinity, chromatography on immobilized EF-Tu·GTP is based on two biochemical selection processes: (1) on aminoacyl-tRNA synthetase catalyzed aminoacylation of tRNA that recognized only its cognate tRNA and the corresponding amino acid, and (2) on recognition of elongation factor Tu-GTP that binds only aminoacyl-tRNA but not tRNA with free 3̒-end. Affinity chromatography in combination with high-performance liquid chromatography (HPLC) analysis of modified nucleosides can efficiently complement the rapid sequencing method and facilitate a complete determination of transfer RNA (tRNA) sequences.
Published Version
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