Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase Journal Article
| Authors: | Jain, R.; Shuman, S. |
| Article Title: | Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3′ terminal RNA ribose 2′O-methyltransferase |
| 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. Published by Cold Spring Harbor Laboratory Press. Copyright © 2011 RNA Society. |
| Keywords: | adomet; rna end-healing; rna repair; transmethylation |
| Journal Title: | RNA |
| Volume: | 17 |
| Issue: | 3 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2011-03-01 |
| Start Page: | 429 |
| End Page: | 438 |
| Language: | English |
| DOI: | 10.1261/rna.2500711 |
| PROVIDER: | scopus |
| PMCID: | PMC3039143 |
| PUBMED: | 21205839 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 4 March 2011" - "CODEN: RNARF" - "Source: Scopus" |
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