Stability and separation of aminoacyl-transfer RNAs on chromatography columns

Abstract

In order to demonstrate that the apparent amount of a tRNA isoacceptor depends on the column matrix employed, chromatographic separations of lysyl tRNAs on several polystyrene anion exchangers and on a reversed-phase matrix (RPC-5) have been studied. Experiments were carried out to distinguish between the actual yield of isoacceptors (aminoacylated and free species) and the apparent yield of isoacceptors (aminoacylated species only). The results indicate that several polystyrene anion exchangers with similar physical properties resolve lysyl tRNAs differently. The differences are noted in the apparent yields and in the degree of chromatographic resolution. When fire column matrices are incubated separately with [ 3H]lysyl tRNAs and the deacylations measured, the results indicate chemical deacylation by two polystyrene anion exchange matrices but not by two other polystyrene matrices or by RPC-5. Further study of two major isoacceptors of lysyl-tRNA on these column matrices confirm that different matrices cause chemical deacylation of the aminoacyl tRNA bond at different rates. Therefore, the apparent yield of an isoacceptor depends upon the column matrix. The dissociation of the ester bond of the aminoacyl tRNA appears to be catalysed by the quaternary ammonium groups of the matrix. Enhanced deesterification of the aminoacyl tRNA bond is noted in slightly alkaline eluants, suggesting a nucleophilic attack by the hydroxyl anion of the medium. The major conclusion to be drawn is that both the yield and relative amounts of isoacceptors are dependent not only upon the resolving power of the column matrix, but also upon the physical and chemical nature of the matrix and upon the experimental conditions employed. This catalytic effect is in addition to the base-catalysed de-esterification promoted by the residual primary, secondary or tertiary amine groups present in the polystyrene anion exchangers.