Biology subjects

Munson, L. M.

Publications and source records attributed to Munson, L. M..

3 recordsLinked to original sources

bioRxiv2026

Mapping the rRNA methylome reveals contributions of methyltransferases to ribosome function and antibiotic sensitivity

Ribosomal RNA (rRNA) folds into a complex structure used as the macromolecular core for protein synthesis. Chemical modification of rRNA contributes to ribosome structure, function, and susceptibility to antibiotics. Despite their importance, the enzymes responsible for specific rRNA modifications remain unknown in most species. In this work, we integrate genetics and biochemistry with sequencing and mass spectrometry to uncover enzymes responsible for rRNA methylation events in Bacillus subtilis. We characterize 17 enzymes responsible for 20 methylation modifications on the 16S and 23S rRNAs, 11 of which are encoded by previously uncharacterized genes. For each rRNA methyltransferase, we define the modification identity, location, and we determine the impact of loss of cognate rRNA methylation on ribosome biogenesis and antibiotic sensitivity. Our findings demonstrate that loss of nearly half of the 17 genes studied results in alterations to ribosome assembly or antibiotic sensitivity underscoring the importance of chemical modifications to ribosome function.

biochemistry↗

bioRxiv2025

TlyA is a 23S and 16S 2'-O-methylcytidine methyltransferase important for ribosome assembly in Bacillus subtilis

Ribosomal RNA (rRNA) is methylated in organisms ranging from bacteria to metazoans. Despite the pervasiveness of rRNA methylation in biology, the function of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2'-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside antibiotics that target the 16S rRNA. We show that{Delta} tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach and based on sequence alignments with other rRNA methyltransferases we tested the importance of potential catalytic residues and S-adenosyl-L-methionine (SAM) cofactor binding sites. We show that TlyA shares the common rRNA methyltransferase catalytic triad KDK and a SAM binding motif GxSxG which differs from Mtb TlyA. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since E. coli lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between B. subtilis, Mtb and more divergent Gram-negative bacteria providing new insight into translation and antibiotic resistance mechanisms.

microbiology↗

bioRxiv2024

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 [A], in what appears to be an inactive state. In this conformation, a predicted -helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg2+-dependent 3-5 DNA exonuclease. We show that Leu113 aids in coordinating the 3 end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/580553v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@ac964borg.highwire.dtl.DTLVardef@bf1f81org.highwire.dtl.DTLVardef@1ad4e89org.highwire.dtl.DTLVardef@ff0641_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗