Biology subjects

Comstock, L. R.

Publications and source records attributed to Comstock, L. R..

4 recordsLinked to original sources

bioRxiv2025

Molecular basis of tRNA substrate recognition and modification by the atypical SPOUT methyltransferase Trm10

The evolutionarily conserved methyltransferase Trm10 catalyzes N1 methylation of guanosine 9 (G9) in select tRNAs, but the basis of specific substrate recognition and modification has remained unclear. Using an S-adenosyl-L-methionine analog, we trapped a post-catalytic state of the Trm10-tRNAGly complex for structure determination by cryogenic electron microscopy. Three distinct complexes were captured: two monomeric Trm10-tRNA complexes with distinct tRNA acceptor stem orientations ("closed" and "open"), and a minor dimeric complex with two Trm10s bound to the same tRNA. The monomeric structures identify conserved residues involved in tRNA interactions across a positively charged surface that guide G9 into the catalytic site and stabilize the flipped nucleotide. In the tRNAopen conformation, acceptor stem rotation weakens tRNA-protein contacts, consistent with a product-release state. The dimeric complex, supported by tRNA-dependent protein crosslinking and molecular dynamics (MD) simulations, positions one Trm10 on G9 similarly to the monomeric complexes, while the other contacts distal tRNA regions, suggesting a functional role in promoting functionally critical conformational transition(s). MD simulations also show how Trm10 achieves selective stabilization of G9 over A9 in the binding pocket. Overall, our findings reveal the mechanism of G9-specific tRNA methylation by Trm10 and suggest a unique mechanism of action among RNA-modifying SPOUT methyltransferases.

biochemistry↗

bioRxiv2025

Mechanism of 30S subunit recognition and modification by the conserved bacterial ribosomal RNA methyltransferase RsmI

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 is able to recognize its 30S substrate and specifically modify its buried target nucleotide. We determined a 2.42 [A] 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 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 activity that suggests RsmI may be first 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 extensive unfolding of a critical rRNA functional center point to a potential role in accurate 30S subunit biogenesis.

biochemistry↗

bioRxiv2023

30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine (SAM) analog to trap the complex in a post-catalytic state to enable determination of an overall 3.0 [A] cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to re-sensitize bacterial pathogens to aminoglycosides. SignificanceIncreasing prevalence of bacterial antibiotic resistance threatens our ability to treat bacterial infections and with it, many other facets of modern healthcare. For the ribosome-targeting aminoglycoside antibiotics, diverse pathogenic bacteria have acquired ribosomal RNA (rRNA) methyltransferase enzymes that confer exceptionally high-level resistance through site-specific modification of the drug binding site. Here, we define the molecular basis for ribosomal substrate recognition and modification by an enzyme (RmtC) representing the most clinically prevalent methyltransferase family. Specifically, RmtC exploits a conserved rRNA surface for binding and induces significant disruption of the rRNA structure to capture the target nucleotide for modification via a "base flipping" mechanism. These insights also present a platform for methyltransferase inhibitor development to extend usefulness of aminoglycoside antibiotics.

biochemistry↗

bioRxiv2021

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

Changes in bacterial ribosomal RNA methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a post-catalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144 which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues. Significance StatementThe bacterial ribosome is an important target for antibiotics used to treat infection. However, resistance to these essential drugs can arise through changes in ribosomal RNA (rRNA) modification patterns through the action of intrinsic or acquired rRNA methyltransferase enzymes. How these antibiotic resistance-associated enzymes recognize their ribosomal targets for site-specific modification is currently not well defined. Here, we uncover the molecular basis for large ribosomal (50S) subunit substrate recognition and modification by the Mycobacterium tuberculosis methyltransferase TlyA, necessary for optimal activity of the antitubercular drug capreomycin. From this work, recognition of complex rRNA structures distant from the site of modification and "flipping" of the target nucleotide base both emerge as general themes in ribosome recognition for bacterial rRNA modifying enzymes.

biochemistry↗