A Divalent Metal Ion-Dependent N 1 -Methyl Transfer to G37-tRNA

Abstract

The catalytic mechanism of the majority of S-adenosyl methionine (AdoMet)-dependent methyl transferases requires no divalent metal ions. Here we report that methyl transfer from AdoMet to N1 of G37-tRNA, catalyzed by the bacterial TrmD enzyme, is strongly dependent on divalent metal ions and that Mg2+ is the most physiologically relevant. Kinetic isotope analysis, metal rescue, and spectroscopic measurements indicate that Mg2+ is not involved in substrate binding, but in promoting methyl transfer. Based on the pH-activity profile indicating one proton transfer during the TrmD reaction, we propose a catalytic mechanism in which the role of Mg2+ is to help to increase the nucleophilicity of N1 of G37 and stabilize the negative developing charge on O6 during attack on the methyl sulfonium of AdoMet. This work demonstrates how Mg2+ contributes to the catalysis of AdoMet-dependent methyl transfer in one of the most crucial post-transcriptional modifications to tRNA.

Mg 2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide

“…At high intracellular [Mg 2+ ], translation of mgtL is rapid and complete (inset 2b), facilitating stem loop B formation, which exposes the rut site and leads to Rho binding and transcription termination, turning mgtA OFF (inset 3b). EF-P, which assists the incorporation of Pro residues into nascent proteins (16) and TrmD, which catalyzes Mg 2+ -dependent m 1 G37 methylation of all three tRNA Pro species (26), are required for rapid translation of mgtL (inset 4). Nucleotide changes in red denote mutations that repress mgtA expression, and nucleotide changes in green denote mutations that turn on mgtA expression.…”

“…Without this methylation, translation of Pro codons is prone to +1-frameshifting (25). TrmD is unique among AdoMet-dependent nucleic acid methyltransferases in that it requires Mg 2+ for its activity (26). TrmD is essential for viability, but the heat-sensitive trmD27 (S88L) allele (27) enabled us to test whether reduction in the levels of m 1 G37-tRNA Pro would decrease the efficiency of translation of the Pro codons of mgtL.…”

A novelHSD17B10mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression

“…[40][41][42][43][44][45][46][47] The transcript of human mttRNA Leu (UUR) was used as the substrate for the synthesis of m 1 G9. The radioactive methyl donor was purchased from Perkin-Elmer.…”

Structural basis for methyl-donor–dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD

“…In the resultant intermediate state, the developing negative charge on O 6 of G37 is stabilized by the positively charged guanidine group of the strictly conserved Arg154*. In a recent study, a Mg 2+ ion was found to stabilize the developing negative charge on O 6 of G37 after proton abstraction, thus contributing to the catalytic mechanism of methyl transfer (19). In the mF o -DF c map of the tRNA•sinefungin-bound form, a weak electron density was identified near N 7 of G37, which might be a partially bound Mg 2+ ion (Fig.…”