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. 2018 Sep 28;46(17):9160-9169.
doi: 10.1093/nar/gky592.

Identification of a novel tRNA wobble uridine modifying activity in the biosynthesis of 5-methoxyuridine

Huijeong Ryu  1 , Tyler L Grove  2 , Steven C Almo  2 , Jungwook Kim  1
Affiliations

Identification of a novel tRNA wobble uridine modifying activity in the biosynthesis of 5-methoxyuridine

Huijeong Ryu et al. Nucleic Acids Res. .

Abstract

Derivatives of 5-hydroxyuridine (ho5U), such as 5-methoxyuridine (mo5U) and 5-oxyacetyluridine (cmo5U), are ubiquitous modifications of the wobble position of bacterial tRNA that are believed to enhance translational fidelity by the ribosome. In gram-negative bacteria, the last step in the biosynthesis of cmo5U from ho5U involves the unique metabolite carboxy S-adenosylmethionine (Cx-SAM) and the carboxymethyl transferase CmoB. However, the equivalent position in the tRNA of Gram-positive bacteria is instead mo5U, where the methyl group is derived from SAM and installed by an unknown methyltransferase. By utilizing a cmoB-deficient strain of Escherichia coli as a host and assaying for the formation of mo5U in total RNA isolates with methyltransferases of unknown function from Bacillus subtilis, we found that this modification is installed by the enzyme TrmR (formerly known as YrrM). Furthermore, X-ray crystal structures of TrmR with and without the anticodon stemloop of tRNAAla have been determined, which provide insight into both sequence and structure specificity in the interactions of TrmR with tRNA.

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Figures

Figure 1.
Figure 1.
Examples of the wobble uridine modification at C5. Chemical structures of modified uridine at the wobble position (U34) is shown with (A) alkylation or (B) O-alkylation at C5 of pyrimidine. 5-methyluridine (m5U), 5-taurinomethyluridine (τm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-hydroxyuridine (ho5U), 5-methoxyuridine (mo5U), 5-carboxymethyluridine (cmo5U), 5-carboxymethoxyuridine (mcmo5U).
Figure 2.
Figure 2.
Proposed biosynthetic pathways for the synthesis of cmo5U and mo5U. (A) In Gram-negative bacteria, Cx-SAM is synthesized from SAM and prephenate by CmoA. Subsequently, CmoB directs carboxymethyltransferation at the 5-hydroxyl group of ho5U34, which is generated by an unknown pathway. (B) TrmR transfers the methyl group from SAM onto ho5U34 of equivalent isoacceptors in Gram-positives. B denotes a general base.
Figure 3.
Figure 3.
Biosynthesis of mo5U by yrrM (or trmR). Presence of mo5U in total RNA extracted from Bacillus subtilis and the single knockout mutants was examined by triple-quadruple (QqQ) mass spectrometry in positive ion mode. Multiple reaction monitoring of mo5U is quantified by tracing the fragmentation of 5-methoxyuracil (mass-to-charge ratio 143.046), and an exemplary chromatogram of the parent strain is displayed (left), whereas no equivalent transition was observed in the sample prepared from ΔyrrM, or ΔtrmR.
Figure 4.
Figure 4.
In vitro mo5U forming activity of TrmR. (A) On the left, HPLC analysis of TrmR directed the formation of mo5U (solid line) versus control which contains no TrmR (dashed line). The peak corresponding mo5U eluted from the C-18 column at 8.6 min which has been confirmed by spiking with an authentic compound (right panel). (B) The assay sample was analyzed by LC-MS/MS in positive mode, where the peak corresponding to mo5U ([C10H15N2O7]+, calculated m/z = 275.088) was selected (left) and the subsequent fragmentation by ESI was confirmed as detection of 5-methoxyuracil ([C5H7N2O3]+, calculated m/z = 143.046) in MS/MS (right).
Figure 5.
Figure 5.
Overall structures of TrmR. The ribbon presentation of the crystal structure of (A) RNA-free TrmR (navy) with SAH (gray stick) and magnesium ion (sphere, magenta), and (B) TrmR (green) bound to tRNAASL (red stick) with SAM (gray stick). (C) Overlaid structures of RNA-free TrmR (blue) and TrmR:tRNAASL (green and red). (D) Ionic/hydrogen bond interactions between ASL tRNA and a set of basic residues of TrmR in the crystal structure of TrmR–ASLAla complex. The ASL is shown with their carbon atoms colored gray, nitrogen atoms colored blue, oxygen atoms colored red and phosphorous atoms colored orange. Hydrogen bond is indicated by dashed lines.
Figure 6.
Figure 6.
Ligand/metal binding by TrmR. (A) Close-up view of SAM-binding site in the crystal structure of Bacillus subtilis. SAM is shown in dark gray. TrmR amino acid residues that interact with SAM are shown in green. Hydrogen bonds are shown as dashed lines. (B) TrmR model colored according to the distribution of electrostatic potential, from red (−5 kT) to blue (5 kT). The SAM-binding pocket is largely negatively charged. (C) Coordination of magnesium ion in RNA-free TrmR structure, where the metal ion (magenta) is coordinated by six ligands; i.e. Asp-133, Asp-159 and Asn-160 residues and three ordered water molecules. (D) An absence of magnesium ion in TrmR:tRNAASL structure. Note Asn160 is in two rotameric states.
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