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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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Overall structure of E. coli RlmJ. The MTase domain is shown in blue and the HS in orange. (A) Domain organization of RlmJ. (B) Topology diagram. β-strands are shown as triangles, α-helices are shown as large circles, and 310-helices are shown as small circles. Dotted circles indicate helices formed on binding of cofactor and substrate (see Figure 4B). (C) Cartoon representation shown in side and front view. A red asterisk indicates the substrate binding site.
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gkt719-F2: Overall structure of E. coli RlmJ. The MTase domain is shown in blue and the HS in orange. (A) Domain organization of RlmJ. (B) Topology diagram. β-strands are shown as triangles, α-helices are shown as large circles, and 310-helices are shown as small circles. Dotted circles indicate helices formed on binding of cofactor and substrate (see Figure 4B). (C) Cartoon representation shown in side and front view. A red asterisk indicates the substrate binding site.

Mentions: The RlmJ structure consists of a discontinuous MTase domain (residues 1–46 and 99–280), interrupted by a helical subdomain (HS, residues 47–98) (Figure 2A and B). Together, they form a compact and globular 40.5 × 37.5 × 47.5 Å3 structure with a prominent pocket on one side (Figure 2C). The MTase domain of RlmJ consists of a central twisted eight-stranded β-sheet flanked by three α-helices on one side and four α-helices on the other side. The first six strands of the β-sheet are parallel, and the last two strands are antiparallel. An extra helix α9 and strand β10 at the C-terminal end of RlmJ distinguishes it from the canonical class I Rossmann-like MTase fold (28).Figure 2.


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)

Overall structure of E. coli RlmJ. The MTase domain is shown in blue and the HS in orange. (A) Domain organization of RlmJ. (B) Topology diagram. β-strands are shown as triangles, α-helices are shown as large circles, and 310-helices are shown as small circles. Dotted circles indicate helices formed on binding of cofactor and substrate (see Figure 4B). (C) Cartoon representation shown in side and front view. A red asterisk indicates the substrate binding site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt719-F2: Overall structure of E. coli RlmJ. The MTase domain is shown in blue and the HS in orange. (A) Domain organization of RlmJ. (B) Topology diagram. β-strands are shown as triangles, α-helices are shown as large circles, and 310-helices are shown as small circles. Dotted circles indicate helices formed on binding of cofactor and substrate (see Figure 4B). (C) Cartoon representation shown in side and front view. A red asterisk indicates the substrate binding site.
Mentions: The RlmJ structure consists of a discontinuous MTase domain (residues 1–46 and 99–280), interrupted by a helical subdomain (HS, residues 47–98) (Figure 2A and B). Together, they form a compact and globular 40.5 × 37.5 × 47.5 Å3 structure with a prominent pocket on one side (Figure 2C). The MTase domain of RlmJ consists of a central twisted eight-stranded β-sheet flanked by three α-helices on one side and four α-helices on the other side. The first six strands of the β-sheet are parallel, and the last two strands are antiparallel. An extra helix α9 and strand β10 at the C-terminal end of RlmJ distinguishes it from the canonical class I Rossmann-like MTase fold (28).Figure 2.

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