Purification and characterization of a Saccharomyces cerevisiae exoribonuclease which yields 5'-mononucleotides by a 5' leads to 3' mode of hydrolysis.
An exoribonuclease producing 5'-mononucleotides has been purified from ribosomes of Saccharomyces cerevisiae. The enzyme has a broad pH optimum around 8.0, requires divalent cation, and is stimulated by monovalent cation with the cation and degree of stimulation being dependent on the substrate used. With either poly(A) or rRNA as substrate, the enzyme has a processive mode of hydrolysis. The oligonucleotides, (pA)3-5, are hydrolyzed by the enzyme, and the hydrolysis is dependent on a 5'-phosphate end group. Phosphorylation of the 3' end has little effect on the rate of hydrolysis. With [3H]poly(A) or [3H]rRNA, labeled differentially at the 5' termini, a more rapid release of 5'-terminal label can be shown, providing evidence that the enzyme hydrolyzes in a 5' leads to 3' direction. Further evidence for a 5' leads to 3' mode of hydrolysis is provided by a study of the products of the hydrolysis of [3H](pA)5 labeled at the 5' termini with 32P. No 32P label is found in (pA)2 which accumulates as an intermediate.
D002413:Cations, Divalent; D002414:Cations, Monovalent; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism*; D005095:Exoribonucleases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009843:Oligoribonucleotides; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D012265:Ribonucleotides / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D002413:Cations, Divalent; D002414:Cations, Monovalent; D009843:Oligoribonucleotides; D012265:Ribonucleotides; D005092:Exonucleases; D005095:Exoribonucleases; D012260:Ribonucleases; C022535:exoribonuclease II