Figure 2

Effect of the putative cofactors on Pa-exosome poly-A RNA degradation activity. (A) RNase activity reactions were performed with 1 pmol radiolabeled 14-mer poly-rA probe incubated with fixed amounts of the indicated purified proteins (100 pmol of either Pa1135 or PaNip7, or 200 pmol of PaSBDS), and varying amounts of the exosome complexes (1, 5 or 10 pmol of either RNase PH ring, PaRrp4-exosome, or PaCsl4-exosome). Lanes 13-15, 10 pmol of exosome complexes. Proteins were incubated with RNA at 65°C for 15 min, in the presence of 10 mM NaH₂PO₄. Samples were separated on 8% denaturing polyacrylamide gel and visualized by phosphorimaging. -, No protein added to the reaction. RNA oligo and degradation products are indicated on the right-hand side. (B-D) Quantitation of bands visualized on denaturing polyacrylamide gels after RNA degradation assay. The ratio of degradation products over substrate RNA in each lane was calculated for the three concentrations of exosome complexes used (1, 5, or 10 pmol), in absence or presence of 100 pmol PaNip7, 200 pmol PaSBDS, or 100 pmol Pa1135. (B) Effect of the three tested proteins on RNase PH ring exosome complex. (C) Effect of the proteins on the PaRrp4-exosome. (D) Effect on the PaCsl4-exosome.