Figure 3.

Effect of oligonucleotides complementary to the 5′ single-strand region of RNA I on PAP I activity. (a) Upper, polyadenylation reactions for RNA I, RNA I + oligo 12 (5′-AAATACTGTCCC-3′), and RNA I + oligo 8 (5′-AAATACTG-3′). Lower, plot of adenylation rates for the three reactions [RNA I (○), RNA I + oligo 12 (■), and RNA I + oligo 8 (⧫)]. (b) RNase E cleavage assays of the substrates shown in a. →, the 8-nt RNase E cleavage product. One picomole of GGG.RNA I 5′-labeled with [γ-³²P]ATP was used in adenylation or RNase E cleavage reactions. RNA I was incubated with either water or 100 pmol of oligonucleotides for 10 min before addition to the reaction mixture. PAP I (280 fmol) was added to each polyadenylation reaction at time 0. RNase E cleavage assay was carried out with 1 μg of recombinant RNase E (53) at 30°C in polyadenylation reaction buffer except that ATP, phosphoenolpyruvate, and pyruvate kinase were omitted.