Purification of potential 3′ processing nucleases using synthetic tRNA precursors

Abstract

The synthetic tRNA precursors, tRNA-C-[¹⁴C]U and tRNA-C-C-A-[¹⁴C]C-C, as well as poly(A) and diesterase-treated tRNA, have been used to identify and purify potential 3′processing nucleases. Four activities have been separated by this analysis; and three of them have been characterized. Two of the enzymes, which are well-separated on hydroxylapatite columns, act on poly(A), require K⁺ and Mg²⁺ for activity, and have molecular weights of about 90,000. These activities have properties previously ascribed to RNase II. The third enzyme does not act on poly(A), requires Mg²⁺ for activity, and has a molecular weight of about 60,000, It is identical to RNase D, previously characterized as an exonuclease acting on tRNAs with altered structure. Each of the enzymes can remove nucleotides from the tRNA precursor containing extra nucleotides beyond the 3′terminus, whereas they are relatively inactive with intact tRNA or tRNA-C-U. The greatest specificity was displayed by RNase D. The possibility that RNase D is a 3′processing nuclease is discussed.