Extended Data Fig. 1: Specificity of Np4N hydrolysis and RNA decapping by E. coli ApaH.
From: ApaH decaps Np4N-capped RNAs in two alternative orientations
(a,b) Representative chromatograms showing the kinetics of Ap4A (a) and Ap4G (b) hydrolysis. (c) Sequences and expected secondary structures of long RNA substrates. Left, a representative RNA substrate bearing two RNA hairpins. RNAs used in the study shared the same stem–loop structures. Right, Ap4A8XL RNA used as an internal standard. This RNA contains three RNA hairpins and a long unpaired 5′ segment. (d) A representative gel showing the kinetics of Ap4G0 RNA decapping. Ap4G0 was mixed with the internal standard Ap4A8XL and treated with ApaH. Decapping of each RNA was monitored as a function of time by boronate gel electrophoresis and fluorescence. (e) Decapping of the invariant internal standard Ap4A8XL (Std). Left to right, decapping of Ap4A8XL in the reactions graphed in Fig. 2a–c, respectively. Time points and error bars represent mean ± s.d. of three independent measurements (n = 3). (f–h) Inhibition of ApaH by reaction products. IC50 values, calculated by fitting the data to the ‘inhibitor vs. response’ model of GraphPad Prism, are indicated for Ap4A + ppAG (f) and Ap4AGG + ppAGG (h). Data points and error bars are mean ± s.d. of three independent measurements (n = 3) for Ap4A+ppAG. Data points for all other reactions are from 2 independent measurements (n = 2). The reactions proceeded for 5 min. (f) Inhibition of Np4A hydrolysis by ppAG, ADP, and UDP. (g) Inhibition of Ap4AG hydrolysis by ADP. (h) Inhibition of Ap4AGG hydrolysis by ppAGG.
