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RNA. 2011 Mar;17(3):429-38. doi: 10.1261/rna.2500711. Epub 2011 Jan 4.

Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3' terminal RNA ribose 2'O-methyltransferase.

Author information

1
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.

Abstract

The RNA methyltransferase Hen1 and the RNA end-healing/sealing enzyme Pnkp comprise an RNA repair system encoded by an operon-like cassette present in bacteria from eight different phyla. Clostridium thermocellum Hen1 (CthHen1) is a manganese-dependent RNA ribose 2'O-methyltransferase that marks the 3' terminal nucleoside of broken RNAs and protects repair junctions from iterative damage by transesterifying endonucleases. Here we used the crystal structure of the homologous plant Hen1 to guide a mutational analysis of CthHen1, the results of which provide new insights to RNA end recognition and catalysis. We illuminated structure-activity relations at eight essential constituents of the active site implicated in binding the 3' dinucleotide of the RNA methyl acceptor (Arg273, Arg414), the manganese cofactor (Glu366, Glu369, His370, His418), and the AdoMet methyl donor (Asp291, Asp316). We investigated the effects of varying the terminal nucleobase, RNA size, RNA content, and RNA secondary structure on methyl acceptor activity. Key findings are as follows. CthHen1 displayed a fourfold preference for guanosine as the terminal nucleoside. RNA size had little impact in the range of 12-24 nucleotides, but activity declined sharply with a 9-mer. CthHen1 was adept at methylating a polynucleotide composed of 23 deoxyribonucleotides and one 3' terminal ribonucleotide, signifying that it has no strict RNA specificity beyond the 3' nucleoside. CthHen1 methylated RNA ends in the context of duplex secondary structures. These properties distinguish bacterial Hen1 from plant and metazoan homologs.

PMID:
21205839
PMCID:
PMC3039143
DOI:
10.1261/rna.2500711
[Indexed for MEDLINE]
Free PMC Article

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