m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

Abstract

A family of RNA m(5)C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m(5)C 967 MTase, Fmu, as an initial probe. The RNA m(5)C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m(5)C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m(5)U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m(5)C MTase uses a different Cys as a catalytic nucleophile than the DNA m(5)C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m(5)C MTases remains unknown.

Scheme 1

Proposed mechanism for methylation of cytosine nucleotides.

Figure 1

SDS/PAGE of Fmu and its complexes with FC RNA. (A) Coomassie-blue-stained gels of Fmu and FC RNA (lane 1); Fmu, FC RNA, and [³H-Me]AdoMet (lane 2); and nuclease P1 digest of covalent complex from lane 2 (lane 3). (B) Autoradiogram of the same gel. MM, molecular mass.

Figure 2

SDS/PAGE of FC RNA complexed to wild-type Fmu (lane 1), Fmu C325A (lane 2), and Fmu C375A (lane 3). Proteins were visualized by staining with Coomassie blue. MM, molecular mass.

Figure 3

Sequence motifs of representative DNA and RNA MTases; bold face residues are conserved in subfamilies. TrmA is m⁵U54 tRNA MTase of E. coli. Fmu-ecoli, Fmu-bacsu, and Fmu-haein are Fmu proteins from E. coli, Bacillus subtilis, and Haemophilus influenzae, respectively. Met2-ecoli and mtbf-bacsu are two DNA m⁵C MTases from E. coli and B. subtilis.