Figure 4.

Reconstitution of CF I polyadenylation activity requires the addition of all five peptides. Whole-cell extract from yeast strain FY86 polyadenylates precleaved precursor before fractionation on a Hi-Trap Q column. Reactions examined in lanes 2–21 were supplemented with the purified recombinant proteins Pab1 and PAP. A fraction containing both CF II and PF I activity can reconstitute polyadenylation activity in the presence of PAP and Pab1 proteins when mixed with either CF I obtained by fractionation of yeast whole-cell extract or all five peptide components of CF I (lanes 4 and 21). The CF I and CF II/PF I fractions are not sufficient for specific polyadenylation in the presence of PAP and Pab1 (lanes 2 and 3).