Open AccessPreparation and Characterization of Fragments from Yeast tRNA phe
Author(s) -
Harbers Klaus,
Thiebe Rainer,
Zachau Hans G.
Publication year - 1972
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1972.tb01749.x
Subject(s) - exonuclease , transfer rna , endonuclease , yeast , ethidium bromide , exonuclease iii , chemistry , cleavage (geology) , oligonucleotide , biochemistry , gel electrophoresis , nucleotide , cyanogen bromide , dna , biology , rna , escherichia coli , peptide sequence , paleontology , fracture (geology) , gene , dna polymerase
The preparation of 13 fragments from yeast tRNA Phe 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 MgCl 2 three regions of tRNA Phe 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 tRNA Phe 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 MgCl 2 . The specificity of the various nucleases towards yeast tRNA Phe is briefly discussed.