FIG. 5.
Adenylylation, U deletion, and U insertion at various PPi concentrations. The purified editing complex was first charged with the indicated amount of [α-32P]ATP (labeled [A] and unlabeled [B and C]) prior to treatment with indicated amounts of PPi plus the other indicated components. (A) Adenylylation assay performed as described for Fig. 2. The similar intensity labeling of DREL and IREL arose because 1 nM ATP charged a small fraction of DREL molecules and a large fraction of available IREL molecules, but most IREL molecules in the purified editing complex were preadenylylated (29). The same result was obtained in the absence of added AMP-CP and UTP (data not shown). (B and C) U deletion and U insertion assays performed as described for Fig. 2. (B) The ratio of partial-to-complete U deletion is similar in all lanes. Unlike the cleavage of U insertion, cleavage of U deletion diminishes at >1 mM PPi (not shown). (D) Quantitation of the deadenylylation of the two ligase proteins shown in panel A and the U deletion and U insertion products from the gels of panels B and C, as in Fig. 2E. Band density is in units of 1,000 pixels (see Materials and Methods).