Abstract

RlmJ catalyzes the m6A2030 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 m6A MTases than to RNA m62A 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.

Highlights

  • Nucleotide methylation is the most frequent type of posttranscriptional modification of rRNA in Escherichia coli ribosomes [1]

  • To obtain a complex of RlmJ with S-adenosylhomocysteine (AdoHcy) and adenosine monophosphate (AMP), i.e. RlmJSAH-AMP, RlmJAPO crystals were soaked in mother liquor containing 5 mM AdoHcy and 20 mM adenosine triphosphate (ATP) for 15 min before cryoprotection

  • The 1.85 A RlmJAPO structure was solved by Molecular replacement (MR) using protein data bank (PDB) entry 2OO3, hypothetical protein LPL1258 from Legionella pneumophila, as a search model

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Summary

Introduction

Nucleotide methylation is the most frequent type of posttranscriptional modification of rRNA in Escherichia coli ribosomes [1]. The 36 rRNA modifications in E. coli cluster around the functional centers of the ribosome: the decoding center, the transfer RNA (tRNA) binding A and P sites, the peptidyl transferase center (PTC) and the peptide exit tunnel. Nucleotide A2030, the modification site of RlmJ, is located in the hairpin loop of helix 72 (H72) at the 50 boundary of domain V in 23S rRNA (Figure 1A). The modification is hidden in the interior of the subunit, agreeing with its appearance at an early stage of 50S assembly [4] and with the observation that RlmJ methylates deproteinized knockout 23S rRNA, but not assembled 50S subunits [2]

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