Fig. 4: Reactivity scope of the Tt preQ1-I riboswitch scaffold.
From: A natural riboswitch scaffold with self-methylation activity
a Anion-exchange HPLC analysis of the preQ1-I RNA-catalyzed reaction with m6preQ1 at 37 °C; 2.5 μM RNA substrate, 225 μM m6preQ1, 2.0 mM MgCl2, pH 6.0. HPLC traces of unmodified RNA, the reacted mixture, and the corresponding m3C-modified synthetic RNA reference is shown for comparison (for reaction time course see Supplementary Fig. 1b). The m3C-modified RNA elutes earlier. Incubation of m3C preQ1-I RNA (isolated from a typical methylation reaction with m6preQ1) under harsh basic conditions produced m3U from m3C under concomitant RNA hydrolysis (for FT-ICR MS characterization of m3U-RNA see Supplementary Fig. 1e); chemical structures of m3C-to-m3U hydrolysis. b preQ1-I RNA reactivity analysis using diverse buffer conditions, nucleobase mutagenesis, atomic mutagenesis, and near-cognate cofactors; bars (gray) show mean ± s.e.m. (n = 3 independent experiments); HPLC traces are depicted in Supplementary Fig. 2, 3; source data are provided in the Source Data file. c The reaction rate is dependent on m6preQ1 concentration. The observed rate constants kobs were determined based on HPLC trace analysis at five concentrations of m6preQ1, ranging from 25 to 300 μM. The red line represents a curve fit to kobs = kmax[m6preQ1]/(Km,app + [m6preQ1]). Individual data points (open circles) (n = 3 independent experiments), mean ± s.e.m. (black circles); source data are provided in the Source Data file. d Chemical structures for c3C and dG used in atomic mutagenesis reactivity assay. e Proposed reaction mechanism of preQ1-I RNA–catalyzed methylation using m6preQ1 as cofactor; key features are the syn-conformation of the methyl iminoester moiety and N1 protonation of m6preQ1; furthermore, coordination of the 2’-OH G11 to O6 of m6preQ1 assists in cofactor alignment and contributes to improve leaving group quality. f UV-spectroscopic determination of the pKa value of m6preQ1 (n = 3 independent experiments); source data are provided in the Source Data file. g Representative isotherm and ‘One set of sites’ bindingÂmodel fit for the preQ1 class I riboswitch titrated with preQ1 (left) and m6preQ1 (right); the indicated Kd values were determined at pH 6 (n = 3 independent experiments); source data are provided in the Source Data file; one experiment for preQ1 is depicted (left): N = 0.97, KA = 2.37 × 107 M−1, ΔH = −27.57 × 103 cal molÂ1, and ΔS = −58.7 cal (mol K)Â1; one experiment for m6preQ1 is depicted (right): N = 0.99, KA = 1.36 × 104 M−1, ΔH = −12.85 × 103 cal molÂ1, and ΔS = −24.2 cal (mol K)Â1. h Chemical structures of other potential cofactors tested.
