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Published on: February 23, 2021
Mechanism of 30S subunit recognition and modification by the conserved bacterial ribosomal RNA methyltransferase RsmI
Mohamed I Barmada1,2, Erin N McGinity1, Suparno Nandi1
1Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
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 C1402, for example, is the only 2'-O-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 recognizes 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 surface previously only seen in the assembled subunit. This positions RsmI to bind a h44 conformation that is substantially reorganized compared to its structure in the mature 30S subunit allowing access to C1402. 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 activity that suggests RsmI may be a member of a distinct class of metal- and SAM-dependent RNA O-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 reorganization of a critical rRNA functional center point to a potential role in accurate 30S subunit biogenesis.
Insights
Bacterial methyltransferase RsmI modifies 16S ribosomal RNA (rRNA) at nucleotide C1402. Cryo-EM reveals RsmI uses a unique binding mode to access its target, impacting ribosome biogenesis and antibiotic susceptibility.
Area of Science:
- Molecular Biology
- Structural Biology
- Microbiology
Background:
- Ribosomal RNA (rRNA) modifications are crucial for ribosome function and bacterial antibiotic resistance.
- The 16S rRNA nucleotide C1402 modification (Cm1402) is essential for peptidyl tRNA binding site structure and is catalyzed by RsmI.
- The mechanism by which RsmI recognizes and modifies its buried target on the 30S ribosomal subunit remains unclear.
Purpose of the Study:
- To elucidate the structural basis and mechanism of RsmI-mediated 16S rRNA modification.
- To understand how RsmI achieves specific recognition and access to the C1402 nucleotide on the 30S ribosomal subunit.
- To investigate the role of RsmI in 30S subunit biogenesis and its implications for antibiotic resistance.
Main Methods:
- Determined the cryo-electron microscopy (cryo-EM) structure of the RsmI-30S complex at 2.42 Å resolution.
- Performed functional analyses to assess the role of RsmI domains and interactions.
- Investigated the requirement for divalent metal ions in RsmI activity.
Main Results:
- RsmI binds the 30S subunit via conserved rRNA surface contacts, utilizing a previously uncharacterized C-terminal domain.
- RsmI induces a reorganized h44 conformation, granting access to the buried C1402 nucleotide.
- RsmI-induced RNA-RNA interactions and a dependence on divalent metal ions suggest a novel class of RNA methyltransferases.
Conclusions:
- The study reveals a unique mechanism for RsmI substrate recognition and modification, expanding understanding of bacterial rRNA methyltransferases.
- RsmI's interaction with distant ribosome features and its role in reorganizing a key functional center suggest involvement in accurate 30S subunit biogenesis.
- Understanding RsmI's mechanism provides insights into bacterial ribosome assembly and potential strategies to modulate antibiotic susceptibility.
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