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Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ.

Punekar AS, Liljeruhm J, Shepherd TR, Forster AC, Selmer M - Nucleic Acids Res. (2013)

Bottom Line: The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI.RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate.RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, PO Box 596, SE 751 24 Uppsala, Sweden.

ABSTRACT
RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

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Binding of substrate-adenosine (pink) to the active sites of m6A DNA MTases and RlmJ. Hydrogen bonds are shown as dotted lines. (A) M.TaqI-DNA complex with the cofactor analogue 5′-deoxy-5′-[2-(amino)ethylthio]adenosine [NEA, PDB 1G38 (37)] (B) T4Dam-DNA complex with the cofactor analogue sinefungin [SFG, PDB 1YFL (41)]. (C) RlmJSAH-AMP with the modeled substrate adenosine (gray) positioned according to superpositioning with M.TaqI.
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gkt719-F5: Binding of substrate-adenosine (pink) to the active sites of m6A DNA MTases and RlmJ. Hydrogen bonds are shown as dotted lines. (A) M.TaqI-DNA complex with the cofactor analogue 5′-deoxy-5′-[2-(amino)ethylthio]adenosine [NEA, PDB 1G38 (37)] (B) T4Dam-DNA complex with the cofactor analogue sinefungin [SFG, PDB 1YFL (41)]. (C) RlmJSAH-AMP with the modeled substrate adenosine (gray) positioned according to superpositioning with M.TaqI.

Mentions: Superposition of structures of DNA m6A MTases in complex with cofactor-like inhibitors and DNA substrate onto RlmJSAH-AMP provides us with a model for binding of accommodated substrate adenosine to RlmJ (Figure 5). The T4Dam complex [PDB 1YFL, (41)] and the M.TaqI complex [PDB 1G38, (37)] superpose on RlmJSAH-AMP with RMSDs of 2.1 Å over 113 Cα atoms and 2.0 Å over 107 Cα atoms, respectively, and the cofactor analogues overlay well.Figure 5.


Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ.

Punekar AS, Liljeruhm J, Shepherd TR, Forster AC, Selmer M - Nucleic Acids Res. (2013)

Binding of substrate-adenosine (pink) to the active sites of m6A DNA MTases and RlmJ. Hydrogen bonds are shown as dotted lines. (A) M.TaqI-DNA complex with the cofactor analogue 5′-deoxy-5′-[2-(amino)ethylthio]adenosine [NEA, PDB 1G38 (37)] (B) T4Dam-DNA complex with the cofactor analogue sinefungin [SFG, PDB 1YFL (41)]. (C) RlmJSAH-AMP with the modeled substrate adenosine (gray) positioned according to superpositioning with M.TaqI.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814359&req=5

gkt719-F5: Binding of substrate-adenosine (pink) to the active sites of m6A DNA MTases and RlmJ. Hydrogen bonds are shown as dotted lines. (A) M.TaqI-DNA complex with the cofactor analogue 5′-deoxy-5′-[2-(amino)ethylthio]adenosine [NEA, PDB 1G38 (37)] (B) T4Dam-DNA complex with the cofactor analogue sinefungin [SFG, PDB 1YFL (41)]. (C) RlmJSAH-AMP with the modeled substrate adenosine (gray) positioned according to superpositioning with M.TaqI.
Mentions: Superposition of structures of DNA m6A MTases in complex with cofactor-like inhibitors and DNA substrate onto RlmJSAH-AMP provides us with a model for binding of accommodated substrate adenosine to RlmJ (Figure 5). The T4Dam complex [PDB 1YFL, (41)] and the M.TaqI complex [PDB 1G38, (37)] superpose on RlmJSAH-AMP with RMSDs of 2.1 Å over 113 Cα atoms and 2.0 Å over 107 Cα atoms, respectively, and the cofactor analogues overlay well.Figure 5.

Bottom Line: The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI.RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate.RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, PO Box 596, SE 751 24 Uppsala, Sweden.

ABSTRACT
RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

Show MeSH
Related in: MedlinePlus