Fig. 2.

Effect of the length of the 5′-terminal single-stranded segment on reactivity with RppH in vitro. (A) Representative gel images. In vitro transcribed A4 and A1 bearing a γ-³²P radiolabel and an internal fluorescein label were mixed with labeled A8XL and treated with purified RppH (8 nM), and the radioactivity (P-32) and fluorescence (Fluor) of each RNA were monitored as a function of time by gel electrophoresis. (B and C) Graphs. RppH-catalyzed phosphate removal from A8, A4, A3, A2, A1, and A1+3 or from G8, G4, G3, G2, G1, and G0 was monitored as in A and quantified by normalizing the radioactivity remaining in each RNA to the corresponding fluorescence intensity. Each time point is the average of two or more independent measurements. Error bars correspond to SDs.