16S rRNA (cytidine1402-2'-O)-methyltransferase / rRNA (cytosine-2'-O-)-methyltransferase activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / four-way junction DNA binding / negative regulation of translational initiation / regulation of mRNA stability ...16S rRNA (cytidine1402-2'-O)-methyltransferase / rRNA (cytosine-2'-O-)-methyltransferase activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / four-way junction DNA binding / negative regulation of translational initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / regulation of DNA-templated transcription elongation / transcription elongation factor complex / DNA endonuclease activity / transcription antitermination / DNA-templated transcription termination / maintenance of translational fidelity / mRNA 5'-UTR binding / regulation of translation / ribosome biogenesis / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / structural constituent of ribosome / ribosome / translation / response to antibiotic / mRNA binding / RNA binding / zinc ion binding / membrane / cytoplasm / cytosol Similarity search - Function
: / RsmI C-terminal HTH domain / SAM-dependent methyltransferase RsmI, conserved site / RsmI AdoMet-dependent methyltransferase protein family signature. / rRNA small subunit methyltransferase I / Tetrapyrrole methylase, subdomain 2 / Tetrapyrrole methylase / Tetrapyrrole (Corrin/Porphyrin) Methylases / Tetrapyrrole methylase, subdomain 1 / Tetrapyrrole methylase superfamily ...: / RsmI C-terminal HTH domain / SAM-dependent methyltransferase RsmI, conserved site / RsmI AdoMet-dependent methyltransferase protein family signature. / rRNA small subunit methyltransferase I / Tetrapyrrole methylase, subdomain 2 / Tetrapyrrole methylase / Tetrapyrrole (Corrin/Porphyrin) Methylases / Tetrapyrrole methylase, subdomain 1 / Tetrapyrrole methylase superfamily / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein S3, bacterial-type / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature. / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein S16 / Ribosomal protein S15, bacterial-type / Ribosomal protein S6 / Ribosomal protein S6 / Ribosomal protein S6 superfamily / Ribosomal protein S12, bacterial-type / Translation elongation factor EF1B/ribosomal protein S6 / Ribosomal protein S18 / Ribosomal protein S18 / Ribosomal protein S18 superfamily / K Homology domain / K homology RNA-binding domain / Ribosomal protein S2 signature 2. / Ribosomal protein S3, conserved site / Ribosomal protein S3 signature. / Ribosomal protein S10, conserved site / Ribosomal protein S10 signature. / Ribosomal protein S14, conserved site / Ribosomal protein S14 signature. / Ribosomal protein S2 signature 1. / KH domain / Type-2 KH domain profile. / K Homology domain, type 2 / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S15/S19, conserved site / Ribosomal protein S19 signature. / Ribosomal protein S10 / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S19/S15 / Ribosomal protein S19/S15, superfamily / Ribosomal protein S19 / Ribosomal protein S2, conserved site / Ribosomal protein S2 / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S2 / Ribosomal protein S7, conserved site / Ribosomal protein S7 signature. / : / K homology domain superfamily, prokaryotic type / Ribosomal protein S5 / S5 double stranded RNA-binding domain profile. / Ribosomal protein S5, N-terminal / Ribosomal protein S5, C-terminal / Ribosomal protein S5, N-terminal domain / Ribosomal protein S5, C-terminal domain / Ribosomal protein S17, conserved site / Ribosomal protein S17 signature. / Ribosomal protein S13, conserved site / Ribosomal protein S13 signature. / Ribosomal protein S13 / 30s ribosomal protein S13, C-terminal / Ribosomal protein S13/S18 / Ribosomal protein S13 family profile. / Ribosomal protein S8 signature. / Ribosomal protein S14 / Ribosomal protein S14p/S29e / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9, N-terminal Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Ribosomal RNA small subunit methyltransferase I / Small ribosomal subunit protein bS21 Similarity search - Component
Biological species
Escherichia coli (E. coli) / Escherichia coli BW25113 (bacteria)
Method
single particle reconstruction / cryo EM / Resolution: 2.42 Å
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
R01 AI088025
United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
F31 AI186518
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
T32 GM135060
United States
Citation
Journal: bioRxiv / Year: 2025 Title: Mechanism of 30S subunit recognition and modification by the conserved bacterial ribosomal RNA methyltransferase RsmI. Authors: Mohamed I Barmada / Erin N McGinity / Suparno Nandi / Debayan Dey / Natalia Zelinskaya / George M Harris / Lindsay R Comstock / Christine M Dunham / Graeme L Conn / Abstract: Ribosomal RNA (rRNA) modifications are important for ribosome function and can influence bacterial susceptibility to ribosome-targeting antibiotics. The universally conserved 16S rRNA nucleotide ...Ribosomal RNA (rRNA) modifications are important for ribosome function and can influence bacterial susceptibility to ribosome-targeting antibiotics. The universally conserved 16S rRNA nucleotide C1402, for example, is the only 2'--methylated nucleotide in the bacterial small (30S) ribosomal subunit and this modification fine tunes the shape and structure of the peptidyl tRNA binding site. The Cm1402 modification is incorporated by the conserved bacterial 16S rRNA methyltransferase RsmI, but it is unclear how RsmI is able to recognize its 30S substrate and specifically modify its buried target nucleotide. We determined a 2.42 Å resolution cryo-EM structure of the RsmI-30S complex and, with accompanying functional analyses, show that RsmI anchors itself to the 30S subunit through multiple contacts with a conserved 16S rRNA tertiary surface present only in the assembled subunit. This positions RsmI to induce an extensive h44 distortion to access C1402 that is unprecedented among 16S rRNA methyltransferases characterized to date. These analyses also reveal an essential contribution to 30S subunit interaction made by the previously structurally uncharacterized RsmI C-terminal domain, RsmI-induced RNA-RNA interactions with C1402, and an unappreciated dependence on a divalent metal ion for catalysis, marking RsmI as the first of a distinct class of metal- and SAM-dependent RNA -methyltransferases. This study significantly expands our mechanistic understanding of how intrinsic bacterial methyltransferases like RsmI modify their rRNA targets. Further, recognition of distant ribosome features and extensive unfolding of a critical rRNA functional center point to a potential role in accurate 30S subunit biogenesis.
Macromolecule #21: Small ribosomal subunit protein bS21
Macromolecule
Name: Small ribosomal subunit protein bS21 / type: protein_or_peptide / ID: 21 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)
Organism: Escherichia coli BW25113 (bacteria)
Molecular weight
Theoretical: 8.524039 KDa
Sequence
String:
MPVIKVRENE PFDVALRRFK RSCEKAGVLA EVRRREFYEK PTTERKRAKA SAVKRHAKKL ARENARRTRL Y
UniProtKB: Small ribosomal subunit protein bS21
+
Macromolecule #22: Ribosomal RNA small subunit methyltransferase I
Macromolecule
Name: Ribosomal RNA small subunit methyltransferase I / type: protein_or_peptide / ID: 22 Details: his-tagged construct with a TEV protease cleavage site preceding the start of the open reading frame with the S'-adenosyl-methionine (SAM) analogue NM6 (PDB code AN6) present in the SAM binding site. Number of copies: 2 / Enantiomer: LEVO / EC number: 16S rRNA (cytidine1402-2'-O)-methyltransferase
pH: 7.6 Details: 10 mM HEPES, 10 mM MgCl2, 100 mM NH4Cl, 6 mM Beta-mercaptoethanol
Grid
Model: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details
Recombinant RsmI (1.2 uM), delta-rsmI 30S (0.4 uM), and the SAM analog NM6 (24 uM) were incubated for 30 minutes at 37C to prepare the complex.
-
Electron microscopy
Microscope
TFS KRIOS
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8164 / Average exposure time: 1.8 sec. / Average electron dose: 49.27 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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