Preparation and characterization of fragments from yeast tRNA phe .

Abstract

The preparation of 13 fragments from yeast tRNAPhe is described. The fragments were prepared by partial digestion with T1-, T2-, N1-, or pancreatic RNAase, by endonuclease and subsequent exonuclease digestion and by a combination of a chemical chain scission with endoor exonuclease degradation. In order to obtain high yields of fragments, in each case several conditions of digestion were investigated by analytical disc electrophoresis. The fragments were purified by column chromatography and preparative gel electrophoresis. They were characterized by their mobility in disc electrophoresis and by analysis of the mono- and oligonucleotides after complete digestion with T1-RNAase. In the presence of MgCl2 three regions of tRNAPhe seem to be particularly exposed to an attack by endonucleases. These regions are the anticodon loop, the dihydrouridine loop and the nucleotides at the 3′-end of the molecule. The T-Ψ-C loop of tRNAPhe seems to be less exposed. Fragments resulting from a cleavage in this region could be obtained in relatively good yield by digestion with N1-RNAase or T1-RNAase, the latter one in the presence of ethidium bromide instead of MgCl2. The specificity of the various nucleases towards yeast tRNAPhe is briefly discussed.