8] The use of methylated albumin silicic acid columns for the fractionation and purification of tRNA

Abstract

This chapter provides an overview of using N-blocked aminoacyl-tRNA derivatives for the purification of amino acid-specific tRNA. The method is relatively easy and rapid, yield is high, and with some modifications, it can be adapted as a general procedure for any tRNA species. However, special precaution are required for highly labile tRNA ester bonds, such as those found with glycyl-, arginyl-tRNAs, and cysteinyl-tRNAs. The procedure in many cases yields a product with a specific activity sufficient for oligonucleotide fingerprint and sequence work. The insignificant level of nonspecific binding of RNA to the methylated albumin adsorbed on a support of silicic acid (MASA) column affords a distinct advantage for these kinds of investigations. The limitations of the system are its dependence on the availability of a purified aminoacyl-tRNA synthetase preparation. Another limitation of the system is that it does not separate isoaccepting tRNAs. By combining this procedure with another separatory technique, such as column chromatography or countercurrent distribution, one can obtain a preparation with a purity of over 90%, and such efforts will be necessary in any case, if different isoaccepting tRNAs are to be separated.