6] The use of BD-cellulose in separating transfer RNA's

Abstract

This chapter describes the use of BD-cellulose in the preparative and analytical fractionation of tRNAs. BD-cellulose is an ion-exchanger with an increased affinity for lipophilic and particularly for aromatic groups. Certain species of tRNA, which contain in their structures a single lipophilic-substituted adenine having cytokinin activity, are retarded relative to most other tRNAs on columns of BD-cellulose, and this is the basis of a simple means of purification of tRNAPhe. Strong salt solution is used to elute all other tRNAs from BD-cellulose, and salt solution containing ethanol or other organic solvent removes the tRNAPhe. The essence of this method is the use of the great specificity of an aminoacyl-tRNA synthetase to attach an aromatic aminoacyl group only to the species of interest in the whole tRNA mixture, and then to utilize the attraction of this aromatic group for BD-cellulose to separate acceptors for this one amino acid from all others. The method described in the chapter is believed to be generally applicable, but quantitative recovery of a given family of isoaccepting tRNAs may not be possible, because of incomplete enzymatic charging, incomplete derivatization, and loss of the amino acid because of the marked lability of the ester bond.