Effect of limiting concentrations of T 1 ribonuclease on the release of aminoacyl-oligonucleotides from unfractionated yeast acceptor RNA

Abstract

Terminal aminoacyl-oligonucleotides are liberated almost quantitatively from yeast sRNA chains charged with either tyrosine, phenylalanine, or glycine by concentrations of T 1 ribonuclease, acting at pH 5·4, 37°C, in the presence of EDTA, that cleave only 17 to 40% of the total GMP sites in the RNA. This suggests that the GMP sites nearest the acceptor end of these chains are more susceptible to the action of T 1 ribonuclease at pH 5·4 than most other GMP sites in the RNA. The first GMP residue in the tyrosine acceptor RNA is in position number 5 while the first GMP residue in the glycine and phenylalanine acceptor RNA is in position number 6. In the case of alanine RNA, where the first GMP residue lies deeper in the chain (position number 8), the preferential release of aminoacyl-oligonucleotide was found to be less pronounced. Substitution of Mg 2+ for EDTA in the digestion mixtures was found to reduce the enzymic release of all the terminal oligonucleotides examined. Studies of the effect of very low levels of T 1 ribonuclease on tyrosine-labeled yeast sRNA at 37 and 58°C in 0·025 m -potassium acetate—0·002 m -EDTA indicate that the most susceptible GMP sites in tyrosine-labeled acceptor chains are deep within the molecule. These results suggest that the GMP site nearest the acceptor end of the molecule is somewhat less susceptible to T 1 enzyme than the above sites but is much more exposed to the enzyme than GMP sites between these two regions. The relevance of these findings to models proposed by other workers for the configuration of sRNA chains is discussed.