Biology subjects

Dziergowska, A.

Publications and source records attributed to Dziergowska, A..

4 recordsLinked to original sources

bioRxiv2026

The Escherichia coli Radical SAM Enzyme YhcC Substitutes for the FAD-Dependent Oxidase Activity of MnmC in 5-Methylaminomethyl-2-Thiouridine tRNA Modification Under Anaerobic Conditions

tRNA wobble uridines are heavily modified to influence anticodon-codon pairing and tune anticodon stem-loop structure for efficient, accurate translation. Many bacteria and some archaea modify wobble uridines with either a 5-carboxymethylaminomethyl (cmnm5) or 5-methylaminomethyl (mnm5) group, often together with a 2-thio (s2) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm5U, which is further converted to mnm5U by non-orthologous enzymes in different lineages. Escherichia coli uses the bifunctional enzyme MnmC to demodify cmnm5U to nm5U and subsequently methylate nm5U to mnm5U whereas Bacillus subtilis relies on the radical SAM (rSAM) enzyme MnmL and the stand-alone methylase MnmM. Although E. coli and related bacteria encode homologs of MnmL, the function of the E. coli homolog, YhcC, remained unknown. Here, we show that YhcC is required for cmnm5s2 U demodification in vivo during anaerobic growth, whereas the equivalent MnmC-dependent reaction requires O2 and occurs only aerobically. In vitro, purified [4Fe-4S]-reconstituted YhcC binds tRNA and catalyzes nm5s2U-tRNA synthesis from cmnm5s2U-tRNA. Together, these results define the previously unknown function of the E. coli rSAM enzyme YhcC and demonstrate that it replaces MnmC under anaerobic conditions to generate nm5s2U. These parallel pathways reveal how E. coli maintains synthesis of a critical wobble-base modification under both aerobic and anaerobic growth conditions.

biochemistry↗

bioRxiv2026

Growth-dependent tRNA Reprogramming and Codon Bias Link Translation to Metabolic State in Enterococcus faecalis

Enterococcus faecalis is a Gram-positive commensal bacterium of the human gut microbiome and an opportunistic pathogen responsible for many hospital-acquired infections. Despite the clinical importance of E. faecalis, how gene and protein expression are coordinated with growth remains poorly defined. Here, we profiled transcript, protein, and tRNA pool dynamics across distinct phases of E. faecalis growth. Differences in protein abundance and corresponding mRNA levels suggested growth phase-dependent posttranscriptional regulation. Growth-associated genes exhibited biased synonymous codon usage, with ribosomal and glycolytic proteins enriched in low-abundance codons read by queuosine-modifiable tRNAs. Analysis of tRNA modification and tRNA isoacceptor abundance revealed growth phase-dependent changes, particularly in anticodon stem loop modifications that influence synonymous codon translation. Changes in queuosine levels preceded shifts in ribosomal proteins, suggesting a contribution to codon-biased translation. Collectively, these findings reveal growth phase-associated remodeling of the E. faecalis tRNA pool and support a model in which queuosine-dependent translational reprogramming shapes protein expression during bacterial growth. IMPORTANCEEnterococcus faecalis is a common cause of hospital-acquired infections. Despite its clinical importance, a comprehensive understanding of the organisms physiology and adaptation to environmental changes remains incomplete. Here, we characterized protein, transcript, and tRNA dynamics across bacterial growth phases, uncovering a role for post-transcriptional regulation marked by tRNA reprogramming and biased synonymous codon usage. These findings enhance our understanding of E. faecalis growth and support a model of translational reprogramming therein.

microbiology↗

bioRxiv2024

Direct RNA Oxford Nanopore sequencing distinguishes between modifications in tRNA at the U34 position

The measurement of tRNA modifications with single transcript resolution has been feasible for only a few modifications due to the lack of available methods. This limitation does not allow to advance basic research studies on the dynamic nature of tRNA modification and its cellular function in time and space, neither to develop modern diagnostic tools for several already known tRNA-dependent human diseases. Nanopore is a well-established sequencing method that has proven to be efficient for the study of RNA. The analysis of tRNA modifications by Nanopore is still under development. We have investigated the efficacy of nanopore technology to discriminate between complex modifications of uridine 34 in tRNA, which affect the base-calling properties of neighbouring bases and are therefore difficult to accurately predict. We have developed new methods to chemically and enzymatically synthesise single modified tRNA molecules with modifications at the anticodon loop. Nanopore technology captures the features produced by uridine with and without a thiol group when present on synthetic tRNA molecules. Thus, Oxford Nanopore Technology (ONT) has great potential for developing strategies to accurately identify the modification status of the tRNA anticodon loop (ACL), which encompasses the most complex modifications on uridine-containing RNA motifs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/630739v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@4317baorg.highwire.dtl.DTLVardef@caf204org.highwire.dtl.DTLVardef@1d7249dorg.highwire.dtl.DTLVardef@1a4f8c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

tRNA modification profiling reveals epitranscriptome regulatory networks in Pseudomonas aeruginosa

Transfer RNA (tRNA) modifications have emerged as critical posttranscriptional regulators of gene expression affecting diverse biological and disease processes. While there is extensive knowledge about the enzymes installing the dozens of post-transcriptional tRNA modifications - the tRNA epitranscriptome - very little is known about how metabolic, signaling, and other networks integrate to regulate tRNA modification levels. Here we took a comprehensive first step at understanding epitranscriptome regulatory networks by developing a high-throughput tRNA isolation and mass spectrometry-based modification profiling platform and applying it to a Pseudomonas aeruginosa transposon insertion mutant library comprising 5,746 strains. Analysis of >200,000 tRNA modification data points validated the annotations of predicted tRNA modification genes, uncovered novel tRNA-modifying enzymes, and revealed tRNA modification regulatory networks in P. aeruginosa. Platform adaptation for RNA-seq library preparation would complement epitranscriptome studies, while application to human cell and mouse tissue demonstrates its utility for biomarker and drug discovery and development.

genetics↗