Bacterial Hen1 is a 3′ terminal RNA ribose 2′-O-methyltransferase component of a bacterial RNA repair cassette

Clostridium thermocellum Hen1. (A) The adjacent co-oriented ORFs encoding the Hen1 (465-amino acid) and Pnkp (870-amino acid) polypeptides comprise a putative two-gene operon. Pnkp is a trifunctional RNA repair enzyme composed of 5′ OH polynucleotide kinase, 2′,3′ phosphoesterase, and ligase-like adenylyltransferase domains. Hen1 is composed of an N-terminal domain unique to the bacterial Hen1 clade and a C-terminal methyltransferase domain (MTase) that is homologous to the methyltransferase domains of eukaryal Hen1 RNA methyltransferase enzymes. The amino acid sequences of the Clostridium thermocellum (Cth), Arabidopsis thaliana (Ath), and Drosophila melanogaster (Dme) Hen1 methyltransferase domains are aligned. (•) Positions of side-chain identity/similarity in all three proteins. (–) Gaps in the alignment. The conserved AdoMet-binding motifs are shaded in gray. (|) The Asp291 and Asp316 residues that were mutated to alanine. (B) CthHen1 purification and methyltransferase activity. (Left) Aliquots (5 μg) of recombinant full-length wild-type (WT) CthHen1, mutants D291A and D316A, and the N-terminal (1–258) and C-terminal (259–465) polypeptide fragments were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kilodalton) of marker polypeptides are indicated on the left. (Right) Reaction mixtures (10 μL) containing 25 mM Tris-HCl (pH 8.5), 0.5 mM MnCl₂, 20 μM [³H-CH₃]AdoMet, 10 μM 24-mer RNA, and 4 μM WT or mutant CthHen1 as specified were incubated for 30 min at 45°C. The extents of ³H-methyl transfer to RNA are plotted.