Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m⁵C methyltransferase

Abstract

Sequence comparison of several RNA m 5 C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m 5 C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m 5 C967 methyltransferase, but whether this conclusion applies to other RNA m 5 C methyltransferases remains to be verified. Yeast tRNA m 5 C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex.

Insights into Folate/FAD-dependent tRNA Methyltransferase Mechanism

“…To examine whether TrmFO BS uses a nucleophilic mechanism, we used an analog of the 31-mer B. subtilis mini-tRNA Asp substrate containing 5-fluorouridine at the target position. Wild-type TrmFO BS was able to form a covalent dihydrouridine intermediate even in the absence of added CH 2 THF but in low amounts compared with other 5-pyrimidine-methylating enzymes examined so far (9,10,12). This result is consistent with the presence of a catalytically competent intermediate containing CH 2 THF, which was estimated to represent at least 17% of the purified protein (4), and indicates a covalent mechanism for tRNA methylation by TrmFO, such as in thymidylate synthase ThyA (15) but in contrast to the most recent mechanism proposed for ThyX (18).…”

“…5-Fluoropyrimidine-containing mini-RNA substrates were judiciously employed as a strategy to probe, trap (9,10,12), and sometimes crystallize (25) these covalent protein-RNA intermediates. Similarly, the formation of a 5,6-dihydropyrimidine intermediate resulting from covalent catalysis in TrmFO BS was ascertained using a 31-mer B. subtilis mini-tRNA Asp containing 5-fluorouridine at the target position (5-FU-mini-RNA; Fig.…”

Human DNMT2 methylates tRNAAsp molecules using a DNA methyltransferase-like catalytic mechanism

“…The apparent equilibrium binding constant of the wild-type DNMT2 was 6 3 10 5 M À1 (Table 1). This value is low, when compared with nucleic acid interaction of other DNA or RNA modifying enzymes (for example, the K Ass for M.EcoRV binding to 40 bp DNA substrate is 1.2 3 10 6 M À1 [Beck et al 2001], and the K Ass for Trm4p binding to tRNA Phe is 2.2 3 10 7 M À1 [Walbott et al 2007]). The weak tRNA binding of DNMT2 may be explained by the fact that the substrate used in the experiment was an in vitro transcribed tRNA Asp , which lacks additional modifications like the mannosylqueosine base (manQ) present at the wobble position (34) of endogenous tRNA Asp (Kuchino et al 1981;Johnson et al 1985).…”

The human tRNA m5C methyltransferase Misu is multisite-specific

“…10 In Saccharomyces cerevisiae, m 5 C formation is catalyzed by a multisite-specific tRNA m 5 C methyltransferase (MTase), Trm4, which uses S-adenosyl-l-methionine (SAM) as methyl donor. [11][12][13] Depending on the tRNA substrate, the target cytosines of Trm4 are located at positions 34, 40, 48 and/or 49. In eukaryotic cells, some of the pre-tRNAs contain introns that are located one nucleotide downstream from the anticodon and are spliced to form the functional tRNA molecule.…”