Figure 5.

Analysis of the ATPase activity of Prp43p variants. ATP hydrolysis time courses were performed with the indicated proteins either in the absence of RNA (A), in the presence of RNA (B) or in the absence of RNA and presence of Pfa1p (C). Wild-type or Prp43p variants at 100 nM were incubated for the indicated times with cold ATP at 100 μM, mixed with α-³²P-ATP tracer. In addition, total yeast RNA was added at 150 μM in (B) and Pfa1p at 500 nM in (C). Aliquots of reaction mixes were then subjected to thin layer chromatography. ATP and ADP were quantified by phosphorimager to derive ADP concentrations. The abbreviations 664, 704, 708, ΔL12, ΔL45 and 1–657 refer to Prp43pR664A-His, Prp43pK704A-His, Prp43pR708A-His, Prp43pΔL12-His, Prp43pΔL45-His and Prp43p(1–657)-His, respectively.