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Synergistic effects of tRNA modification defects in Escherichia coli K12

ABSTRACT: Transfer RNAs (tRNAs) are essential components of the translation machinery and carry numerous post‑transcriptional modifications that contribute to decoding accuracy, efficiency and cellular fitness. In Escherichia coli K‑12, all tRNA modification pathways have been identified, yet the functional interactions between these pathways remain largely unexplored. Here, we systematically analyses genetic interactions between 29 non‑essential tRNA modification genes using a pairwise synthetic lethal screen based on P1 transduction. Most combinations of tRNA modification gene deletions are tolerated during growth in rich medium; however, we identify five synthetically lethal pairs and fifteen additional combinations that display negative genetic interactions. Deletions of truA, which encodes the pseudouridine synthase responsible for modifications at positions 38–40 of multiple tRNAs, show the highest frequency of negative epistasis. Synthetic lethality associated with truA can be complemented by expression of truA in trans and, in specific cases, partially suppressed by overexpression of its tRNA substrates, indicating substrate‑specific functional dependencies. Analysis of tRNA abundance by northern blotting and AQRNA‑seq demonstrates that loss of individual tRNA modification enzymes does not generally lead to widespread tRNA destabilization. Instead, further phenotypic characterization of viable double mutants reveals condition‑dependent growth defects influenced by carbon source, temperature and metabolic stress, as well as toxicity associated with overexpression of specific tRNAs. Together, these results reveal a limited but distinct set of genetic interactions among bacterial tRNA modification pathways and highlight the importance of physiological context in uncovering their cellular roles.

ORGANISM(S): Escherichia coli

PROVIDER: GSE331519 | GEO | 2026/05/20

REPOSITORIES: GEO

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Project description:Transcriptional repression of ribosomal components and tRNAs is coordinately regulated in response to a wide variety of environmental stresses. Part of this response involves the convergence of different nutritional and stress signaling pathways on Maf1, a protein that is essential for repressing transcription by RNA polymerase (pol) III in Saccharomyces cerevisiae. Here we identify the functions buffering yeast cells that are unable to down-regulate transcription by RNA pol III. MAF1 genetic interactions identified in screens of non-essential gene-deletions and conditionally-expressed essential genes reveal a highly interconnected network of 64 genes involved in ribosome biogenesis, RNA pol II transcription, tRNA modification, ubiquitin-dependent proteolysis and other processes. A survey of non-essential MAF1 synthetic sick/lethal (SSL) genes identified six gene-deletions that are defective in transcriptional repression of ribosomal protein (RP) genes following rapamycin treatment. This subset of MAF1 SSL genes included MED20 which encodes a head module subunit of the RNA pol II Mediator complex. Genetic interactions between MAF1 and subunits in each structural module of Mediator were investigated to examine the functional relationship between these transcriptional regulators. Gene expression profiling identified a prominent and highly selective role for Med20 in the repression of RP gene transcription following treatments with rapamycin, chlorpromazine and tunicamycin and in post-diauxic cells. In addition, attenuated repression of RP genes by rapamycin was observed in a strain deleted for the Mediator tail module subunit Med16. The data suggest that Mediator and Maf1 function in parallel pathways to negatively regulate RP mRNA and tRNA synthesis. Keywords: genetic modification, stress response We generated 12 microarray profiles from fluor-reversed replicates of wild-type and med20? strains with or without treatment with rapamycin and under 5 other conditions that repress ribosomal protein gene transcription. The effect of rapamycin on strains deleted for 3 other Mediator subunits was also assessed relative to wild-type. See Willis et al., (2008) in revision.

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Synergistic effects of tRNA modification defects in Escherichia coli K12

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