Alternative RNA degradation pathways by the exonuclease Pop2p from Saccharomyces cerevisiae

Distinct reaction products during RNA degradation by Pop2p. (A) Reaction scheme for Pop2p exonuclease reaction with addition of new Pop2p. (B) Representative PAGE for an exonuclease reaction with A₃₆ and addition of new Pop2p. Pre-steady-state exonuclease reaction (Pop2p: 1200 nM, A₃₆: 1 nM, aliquots were removed at 1, 5, 10, 20, 30, 40, 50, and 60 min), followed by heat inactivation of Pop2p at 60 min (65°C, 20 min) and addition of fresh Pop2p (1200 nM). Aliquots were removed after addition of fresh Pop2p at 15, 30, 40, 50, and 60 min (middle). (Right) Control reaction without addition of fresh Pop2p after heat inactivation. (C) Reaction scheme for Pop2p exonuclease reaction with terminal reaction products. (D) Representative PAGE for an exonuclease reaction of Pop2p (1200 nM) and Rrp44p (1200 nM) with isolated, terminal reaction products (1 nM). Aliquots were removed at 15, 30, 40, 50, and 60 min. (E) Reaction scheme for generation of the internally radiolabeled RNA and for the Pop2p exonuclease reactions with this substrate. The asterisks represent the radiolabeled ³²P linkages between adenosines. (F) Representative PAGE for exonuclease reactions of Rrp44p (1200 nM) and Pop2p (1200 nM) with internally radiolabeled RNA substrate (aliquots: 15, 30, and 60 min). Products generated after 60 min were treated with Antarctic Phosphatase. (G) Representative TLC for Pop2p and Rrp44p exonuclease reactions with internally radiolabeled RNA substrate (aliquots: 15, 30, and 60 min). Products generated after 60 min were treated with Antarctic Phosphatase. (H) Correlation of the fraction monophosphate (5′-AMP + 3′-AMP vs. unreacted product) generated during the reaction (time points: 15, 30, and 60 min) measured by TLC and PAGE.