Biology subjects

Kuiper, E. G.

Publications and source records attributed to Kuiper, E. G..

3 recordsLinked to original sources

bioRxiv2023

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the 9th nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

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↗

bioRxiv2019

Substrate recognition by the Pseudomonas aeruginosa EF-Tu methyltransferase EftM

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, and severe burns. During infection, P. aeruginosa adhesion to host epithelial cells is enhanced by surface exposed translation elongation factor EF-Tu carrying a Lys5 trimethylation. This modification is incorporated by the S-adenosyl-L-methionine-dependent methyltransferase EftM. Thus, EF-Tu modification by EftM may represent a novel target to restrict the establishment of P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM action by defining the molecular mechanism of EF-Tu substrate recognition by this enzyme. First, following the observation that EftM can bind to EF-Tu lacking an N-terminal peptide (encompassing the Lys5 target site), an EftM homology model was generated and used in protein-protein docking studies to predict EftM:EF-Tu interactions. The predicted protein-protein interface was then experimentally validated using site-directed mutagenesis of residues in both proteins coupled with binding and methyltransferase activity assays. We also show that EftM is unable to methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to allow placement of the first 5-6 amino acids of EF-Tu into the conserved peptide binding channel. In this channel, a group of residues that are highly conserved in EftM family proteins position the N-terminal sequence to facilitate modification of Lys5. Our findings provide detailed insights into substrate recognition by this lysine methyltransferase, paving the way for a deeper understanding of EftMs mechanism of action on EF-Tu.

biochemistry↗