Fig. 5: A proposed catalytic mechanism of MTR1.

a, The chemical mechanism. For clarity, we show the mechanism in three steps. In step 1, the nucleobase of C10 becomes protonated, and in step 2, the O⁶-methylguanine becomes bound. However, it is likely that these two steps are coordinated, as the binding will raise the pK_a of the cytosine. The methyl transfer reaction occurs in step 3 by the nucleophilic attack of A63 N1 on the methyl group of O⁶-methylguanine and the coordinated breakage of the guanine O6–C bond. This involves a train of electron transfers, the movement of the proton from C10 N3 to guanine N1 and concomitant shift of the positive charge from C10 to the N¹-methyladenine at position 63. In principle, the guanine can now be released as product, although there is no evidence that this occurs with the present form of the ribozyme. Regeneration of active ribozyme would also require an exchange of the substrate strand to place unmethylated adenine at position 63. The proposed mechanism is fully consistent with the structure of the MTR1 riboswitch and the effect of the substitutions at C10 and U45 on activity (Fig. 4). The complete loss of methylation activity of the C10U variant is fully consistent with the proposed role as general acid in addition to ligand binding. rib, ribose b, The pH dependence of MTR1-catalyzed alkyl transfer. The rate of alkyl transfer was measured using a fluorescent assay (Extended Data Fig. 7) multiple times at pH values between 5 and 8.5. The mean values of observed rate constants derived from at least four independent experiments are plotted as a function of reaction pH, where the error bars represent s.d. The data have been fitted to two ionizations (Methods), from which apparent pK_a values of 5.0 and 6.2 were calculated. The following are the numbers of independent replicates at each pH: pH 5.0, n = 5; pH 5.5, n = 5; pH 6.0, n = 4; pH 6.5, n = 5; n = 5; pH 7.0, n = 5; pH 7.5, n = 4; pH 8.0, n = 4; pH 8.5, n = 4.