The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

Abstract

Modifying RNA enzymes are highly specific for substrate-rRNA or tRNA-and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m(2)A synthesis at purine 37 in a tRNA set still remains unknown. m(2)A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The ΔrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m(2)A(37) from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the ΔrlmN mutant is due to loss of m(2)A from 23S rRNA and, accordingly, that the m(2)A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.

FIGURE 1.

RlmN is the first example of a methyltransferase working on both rRNA and tRNA. The figure shows the distribution of the Escherichia coli modified nucleotides by RNA type. The position of each modification (in parentheses) and the enzyme(s) responsible for its synthesis are indicated. Only two enzymes, RluA and the herein studied RlmN, have dual substrate specificity, recognizing both rRNA and tRNA. (*) Modifications of 16S rRNA; (unk) unknown enzyme; (^‡) modification h⁵C2501 is partial and dependent on the growth phase (Andersen et al. 2004; Havelund et al. 2011).

FIGURE 2.

Sequence and structural comparisons of the RlmN substrates. (A) Sequence and secondary structure of the peptidyl transferase region of the E. coli 23S rRNA. Modification m²A at position 2503 is highlighted in black. The remaining post-transcriptional modifications in the region are highlighted in gray. Note location of helices 90–92, which are crucial for recognition by RlmN. In the bottom right box, a short multiple sequence alignment (seven positions in length) of 23S rRNA and the tRNAs carrying m²A37 is used to search for the potential identity determinants recognized by the m²A-synthesizing enzyme. Positions at the consensus pattern are represented according to the IUPAC nucleotide code. Positions 35 and 36 of substrate tRNAs showing a significant conservation pattern were chosen herein for subsequent studies. (B) The three-dimensional structure corresponding to 2455–2580 nucleotides of the Escherichia coli 23S rRNA sequence was extracted from the 3OAS structure stored in the PDB database and shown in ribbon representation (left-hand side) using UCSF Chimera viewer (Pettersen et al. 2004). Helices required for optimal RlmN action (Yan et al. 2010) are depicted in color, and the target nucleoside for the RlmN-mediated methylation is highlighted in red. The three-dimensional, global structure of the Escherichia coli tRNA^Gln_CUG (extracted from the 2RE8 PDB record) is shown in ribbon representation on the right-hand side. The D, anticodon, and T stem–loops are depicted in color, resembling the secondary structures shown on the left-hand side. The target nucleoside for RlmN-mediated modification is also highlighted in red.

FIGURE 3.

RlmN modifies both rRNA and tRNA. (A) rRNA/tRNA HPLC-nucleoside profile of the Escherichia coli ΔrlmN mutant. After isolation and P1 digestion, bulk rRNA (left) and tRNA (right) from the wild-type and ΔrlmN strains were separately analyzed by HPLC. Nucleoside m²A was identified by its elution time and UV spectra. Note that nucleoside t⁶A (present at position 37 of some tRNAs that are not RlmN substrates) is unaffected in the ΔrlmN cells. (B) In vivo complementation of the ΔrlmN mutant. Recombinant protein His-RlmN, expressed from pET15b-his-rlmN, restores the m²A synthesis in ΔrlmN cells (right panel). Wild-type and ΔrlmN cells containing pET15b (left and middle panels, respectively) were used as controls. Absorbance was monitored at 260 nm. (mAU) Absorbance units ×10⁻³.

FIGURE 4.

RlmN methylates tRNA^Chimera_UUG both in vivo and in vitro. (A) The sequence and secondary structure of the scaffold tRNA is shown together with a representative section of its HPLC chromatogram (D and Ψ modifications elute at early times and are not included in the figure). (B) The tRNA^Chimera_UUG sequence (red nucleotides indicate the ASL from tRNA^Gln_cmnm5s2UUG) is shown together with a section of the corresponding chromatogram. (C) Synthesis in vivo of m²A on tRNA^Chimera_UUG (left panel) is prevented by mutation A37C (middle panel) or by the expression of the wild-type tRNA^Chimera_UUG in ΔrlmN cells (right panel). (D) In vitro methylation of tRNA^Chimera_UUG purified from ΔrlmN cells is carried out by in vitro-reconstituted RlmN in a SAM-dependent manner (red lines). Reconstituted RlmN does not work on in vitro synthesized tRNA^Gln_UUG (black lines). Solid and discontinuous lines indicate that the modification reactions were performed in the presence and absence of SAM, respectively. Absorbance was monitored at 260 nm. (mAU) Absorbance units ×10⁻³.