Biology subjects

Koutmou, K.

Publications and source records attributed to Koutmou, K..

5 recordsLinked to original sources

bioRxiv2026

Ribosomal Protein bL27 Protects Translating Ribosomes from tmRNA-SmpB

Bacterial ribosomal protein bL27 is universally conserved and its amino terminus is adjacent to the peptidyl transfer center, yet its role in translation remains unclear. Combining genetics, biochemistry and molecular dynamics, we show that bL27 has an unexpected role in preventing trans-translation, the bacterial ribosome rescue mechanism, from interfering with protein synthesis. Deletion of the bL27 gene causes a 10,000-fold decrease in viability and this defect is partially rescued by deletion of the gene encoding tmRNA, a critical molecule for trans-translation. Molecular dynamics simulations also indicate that bL27 can slow the movement of tmRNA on the ribosome. These data link trans-translation and bL27, and support a model in which the amino terminus of bL27 acts as a gatekeeper to prevent tmRNA from sterically interfering with tRNA on the ribosome.

biochemistry↗

bioRxiv2025

tRNA isodecoder analysis using Nanopore ionic current signals and deep learning

tRNA are short non-coding RNA characterized by their distinct tertiary structure and abundant chemical modifications. Conventional analysis strategies do not fully characterize tRNA isodecoders. We demonstrate that this limitation can be resolved for tRNA using nanopore ionic current data. We developed tRNAZAP, a deep learning strategy that uses nanopore ionic current signal information to classify native tRNA strands at isodecoder-level resolution without relying on sequence information. Additionally, the ionic current level classification allows for pairwise alignment of read sequences to reference sequences, producing optimal tRNA alignments. We applied tRNAZAP to direct tRNA sequencing data from Escherichia coli and Saccharomyces cerevisiae, and recovered 2.6% and 13.1% more aligned reads than BWA-MEM, respectively. tRNAZAP resolved these reads at an isodecoder-level and with consistently higher alignment identity. tRNAZAP is a powerful complement to sequence-based profiling and can contribute towards resolving the isodecoder landscape in more complex organisms including humans.

genomics↗

bioRxiv2025

The tRNA dihydrouridine synthase DusA has a distinct mechanism in optimizing tRNAs for translation

Dihydrouridine (D) is one of the most highly conserved RNA modifications across all domains of life. D20 within the tRNA D loop is particularly conserved and is formed by DusA in Escherichia coli. However, the mechanisms and cellular functions of DusA and D20 remain poorly understood. Here, we characterize DusAs role in tRNA binding, cofactor oxidation, and modification activity, along with its impact on tRNA maturation and translation. We find that DusA binds tRNA via a two-step mechanism involving a local structural rearrangement and exhibits a higher affinity for previously modified tRNA compared to unmodified tRNA. Unlike the T arm modifying enzymes TrmA and TruB, DusA does not broadly increase cellular aminoacylation for all tRNAs but enhances the charging of specific tRNA species. Despite limited alterations in overall tRNA charging and abundance in cells lacking DusA, DusA selectively improves translation at several specific codons, suggesting a direct contribution for dihydrouridine to the function of certain tRNAs on the ribosome. In conclusion, our findings suggest DusA acts non-redundantly with and complementary to TrmA and TruB in fine-tuning protein synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/672980v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1191c4forg.highwire.dtl.DTLVardef@191b43borg.highwire.dtl.DTLVardef@cfceeborg.highwire.dtl.DTLVardef@119c26a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

A humanized yeast model reveals dominant-negative properties of neuropathy-associated alanyl-tRNA synthetase mutations

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that ligate tRNA molecules to their cognate amino acids. Heterozygosity for missense variants or small in-frame deletions in five ARS genes causes axonal peripheral neuropathy, a disorder characterized by impaired neuronal function in the distal extremities. These variants reduce enzyme activity without significantly decreasing protein levels and reside in genes encoding homo-dimeric enzymes. These observations raise the possibility of a dominant-negative effect, in which non-functional mutant ARS subunits dimerize with wild-type ARS subunits and reduce overall ARS activity below 50%, breaching a threshold required for peripheral nerve axons. To test for these dominant-negative properties, we developed a humanized yeast assay to co-express pathogenic human alanyl-tRNA synthetase (AARS1) mutations with wild-type human AARS1. We show that multiple loss-of-function, pathogenic AARS1 variants repress yeast growth in the presence of wild-type human AARS1. This growth defect is rescued when these variants are placed in cis with a mutation that reduces dimerization with the wild-type subunit, demonstrating that the interaction between mutant AARS1 and wild-type AARS1 is responsible for the repressed growth. This demonstrates that neuropathy-associated AARS1 variants exert a dominant-negative effect, which supports a common, loss-of-function mechanism for ARS-mediated dominant peripheral neuropathy.

genetics↗

bioRxiv2018

N4-acetylcytidine and 5-formylcytidine are present in Saccharomyces cerevisiae mRNAs

Post-transcriptional nucleoside modifications have long been recognized as key modulators of non-coding RNA structure and function. There is an emerging appreciation that the chemical modification of protein-coding messenger RNAs (mRNAs) also plays critical roles in the cell. Although there are over 100 known RNA modifications found in biology only a handful have been identified in mRNAs. We sought to identify and quantify modifications present in the mRNAs of yeast cells using a high throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that measures the levels of 36 types of RNA nucleosides in parallel. We detected the presence of six modified nucleosides in mRNAs at relatively high abundances: N7-methylguanosine, N6-methyladenosine, 2-O-methylguanosine, 2-O-methylcytosine, N4-acetylcytidine and 5-formylcytidine. Additionally, we investigated how the levels of mRNA modifications vary in response to cellular stress. We find that the concentrations of mRNA modifications including N6-methyladenosine and N4-acetylcytidine change in response to heat stress, glucose starvation and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs, and utilizes a high-throughput approach to search for modifications that highlights the value of integrating mass-spectrometry tools in the mRNA modification discovery and characterization pipeline.

biochemistry↗