Tertiary and secondary structures of the E. coli 23S rRNA domain V and enzymatic formation of 7-methylguanosine (m⁷G2069) and N²-methylguanosine (m²G2445). (A) The tertiary structure of domain V in E. coli 23S rRNA (PDB ID 2aw4). The strands G2061-C2084, G2235-C2258, C2422-D2449 are colored blue, green and red, respectively. Coordinates for Helix 93 are not displayed because it lies in front of Helix 74. The silhouette of 50S subunit was showed in gray line. (B) The secondary structure of domain V in the E. coli 23S rRNA with modified nucleotides, 7-methylguanosine (m⁷G), N²-methylguanosine (m²G), 2′-O-methylguanosine (Gm), dihydrouridine (D), pseudouridine (Ψ), 2′-O-methylcytidine (Cm), 5-hydroxycytidine (ho⁵C), N²-methyladenosine (m²A) and 2′-O-methyluridine (Um). The numbers of the RNA helices and nucleotides are indicated. Watson–Crick-type base pairs are shown by bars, and wobble base pairs by black dots. Helix 74 and surrounding regions are boxed on the left. The color code for each strand is the same as (A). (C) Enzymatic formation of m⁷G2069 and m²G2445 is catalyzed by RlmK and RlmL, respectively, using Ado-Met as a methyl-group donor. Ado-Met is converted to Ado-Hcy (S-adenosylhomocysteine) by the reaction.

ycbY (rlmKL) is responsible for both m⁷G2069 and m²G2445 formation. (A) LC/MS analyses of RNase T1 (left panels) and RNase A (right panels) digests of rRNAs from the wild-type (Top row of panels), genome-deletion strain OCR55 (Second row), ΔycbY (rlmKL) strain (Third row) and ΔycbY (rlmKL) strain carrying pRlmKL (Bottom row). Left and right panels show mass chromatograms detecting the triply-charged ions of the 16-mer fragment bearing m⁷G2069 (m/z 1706) and the doubly charged ions of the hexamer fragment bearing m²G2445 (m/z 1024), respectively. (B) Collision-induced dissociation (CID) spectra of RNA fragments bearing m⁷G2069 (left panel) and m²G2445 (right panel). The six-charged ion (m/z 852.4) of the 16-mer fragment carrying m⁷G2069 and doubly charged ion (m/z 1023.7) of the hexamer fragment carrying m²G2445 were used as parent ions for CID. The sequences were confirmed by assignment of the product ions. The nomenclatures for product ions of nucleic acids are as suggested in the literature (41).

Complementation study of m⁷G2069 and m²G2445 formation by plasmid-encoded RlmKL and mutants. (A) Domain structure of E. coli RlmKL. RlmKL consists of two methyltransferase domains, COG0116 and COG1092. We designated the N-terminal domain (COG0116) as RlmL and the C-terminal domain (COG1092) as RlmK. Each domain has an Ado-Met-dependent methyltransferase motif, UPF0020 in the RlmL domain and Cons_hypoth 95 in the RlmK domain. A THUMP domain was found in the N-terminal region of the RlmL domain. (B) Complementation of the ΔrlmKL strain by introducing plasmid-encoded RlmKL or RlmKL mutants. On the left hand side, the domain structures and the mutation positions of RlmKL are shown. On the right hand side, mass chromatograms detecting the 16-mer fragment carrying m⁷G2069 (m/z 1706) and the hexamer fragment carrying m²G2445 (m/z 1024), respectively, are shown for the ΔrlmKL strain transformed with pRlmKL (top panels), pRlmKL N309A (second panels), pRlmKL N397A (third panels), pRlmK (fourth panels), pRlmKL D568A (fifth panels) and pRlmL (bottom panels). (C) Complementation of the E. coli ΔrlmKL strain by introducing N. meningtidis RlmL (NMB0445) or RlmK (NMB1367). Mass chromatograms on the right panels are the same as in (B).

In vitro reconstitution of m⁷G2069 and m²G2445 formation by RlmKL. (A) SDS–PAGE analysis of the purified recombinant RlmKL, RlmL(NTD) and RlmK(CTD) stained with coomassie Brilliant Blue R-250. (B) In vitro reconstitution of m⁷G2069 and m²G2445 by RlmKL, RlmL(NTD) or RlmK(CTD). Left and right panels show mass chromatograms detecting the triple-charged ions of the 16-mer fragment bearing m⁷G2069 (m/z 1706) and the double-charged ions of the hexamer fragment bearing m²G2445 (m/z 1024), respectively. Conditions for in vitro methylation are indicated in the right hand side. The hexamer fragment with m²G2445 produced by RNase A digestion of domain V is Gm²GGGAUp (m/z 1023, MW 2047). (C) Time-course of m²G2445 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmL(NTD) (open circle), 0.1 µM RlmL(NTD) with 0.1 µM RlmK(CTD) (closed triangle), 0.1 µM RlmL(NTD) with 0.2 µM RlmK(CTD) (open triangle), 0.1 µM RlmL(NTD) with 0.4 µM RlmK(CTD) (closed square), 0.1 µM RlmL(NTD) with 0.8 µM RlmK(CTD) (open square) and 0.1 µM RlmKL (closed circle). (D) Time-course of m⁷G2069 formation in domain V (transcript 1) catalyzed by 0.1 µM RlmK(CTD) (open circle) and 0.1 µM RlmKL (closed circle). (E) Truncated RNA substrates. Open and closed circles in the secondary structures indicate the positions of G2069 and G2445, respectively. Detailed constructs for transcripts 3–7 are shown in Supplementary Figure S3. (F) Truncation of domain V (transcript 1) to identify the minimum substrate. The ratio of methylation (%) was calculated from the area of mass chromatograms for the methylated and unmethylated fragments at the end point of the reaction (see Materials and Methods). White and black bars represent the efficiency of m⁷G2069 and m²G2445 formation, respectively.

Unwinding activity of RlmKL. (A) The RNA substrate used for the unwinding assay. 5′-end of transcript 8 was labeled by ³²P. (B) The unwinding assay by RlmKL. The duplex and single strands were separated by native PAGE and the radio activity of transcript 8 was visualized and quantified by the imaging analyzer. Duplex ratio (%) described at the bottom of the panel stands for the remaining duplex proportion in total radio activity of transcript 8. The left 5 lanes and the right 5 lanes show time course monitoring of the unwinding activity in the presence of RlmKL (RlmKL⁺) and BSA (BSA⁺), respectively. (C) Duplex ratio (%) was plotted against reaction time (min). Open and closed circles indicate BSA⁺ and RlmKL⁺, respectively. The half-lives (t_1/2) for both conditions were calculated by non-linear curve fitting and shown as average ±SD; values of three independent experiments.

Substrate recognition of RlmKL. (A) The secondary structure of transcript 3, which contains base flipping mutation C2096G/G2193C to be cut by MazF at the single position. Closed arrowhead show the cleavage position by MazF. (B) Design of mutants based on the transcript 3. Arrows and boxes indicate the substitutions. (C) Methylation activities of the transcript 3 variants. The left and right graphs show the end point of m⁷G2069 and m²G2445 formation, respectively. The relative activities of methylation (%) were calculated and normalized by wild-type sequence. (D) The recognized residues for m⁷G2069 and m²G2445 formation in transcript 3. The residues whose mutations reduced m⁷G2069 or m²G2445 formation to <50% are depicted as white letters. In the left panel, the residues that are depicted in blue background indicates the m⁷G2069 forming activity. In the right panel, the residues that are depicted in red background indicates the m²G2445 formation activity. Color gradation of blue and red background for each residue represents magnitude of importance for the methylation. (E) Crystal structure of Helix 74 and surrounding region in the E. coli 50S subunit. Coordinates were obtained from PBD (2aw4) (3). We added the methyl groups for m⁷G2069 and m²G2445, shown in red, respectively. The recognized residues for m⁷G2069 and m²G2445 formation are shown in blue and red, respectively.

Synthetic growth phenotype of ΔrlmKL and ribosomal subunit profiling. (A) Exploring the synthetic growth phenotype of ΔrlmKL in the presence of the disruption of RNA helicases, including deaD, rhlE, srmB and dbpA. Each strain was serially diluted (1:10 dilutions), spotted onto LB plates and incubated at 37°C or 28°C for 11 and 22 h, respectively. The doubling time (in minutes) is shown to the right of each panel. (B) SDG profiling of ribosomal subunits in cell lysates in the presence of 0.5 mM Mg²⁺ for the wild-type (blue line), ΔrlmKL (red line), ΔdeaD (red line) and ΔrlmKL/ΔdeaD (red line) strains. The amount of each subunit was quantified by UV absorbance at 254 nm. The peak positions for the 30S and 50S subunits are indicated. The peak height was normalized to the 30S peak.

Schematic depiction of a catalytic mechanism for the cooperative modifications of Helix 74. After transcription of domain V in the 23S rRNA, the C-terminal RlmK domain of RlmKL unwinds Helix 74 upon recognizing Helix 80 and Helix 74, and then methylates G2069. The single-stranded 3′ side of Helix 74 is then recognized by the N-terminal RlmL domain, and G2445 is methylated. Finally, Helix 74 reforms a duplex, and domain V is restructured.