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Related Concept Videos

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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The DNA replication, transcription, and translation processes are intricately coupled in bacteria, allowing efficient gene expression and rapid protein synthesis. While this physical and functional coordination is advantageous, it introduces challenges that bacteria overcome through specific regulatory mechanisms.Coupling of Replication, Transcription, and TranslationThe coupling of replication, transcription, and translation is a hallmark of bacterial gene expression. As the replisome unwinds...
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
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Bacterial growth is closely tied to nutrient availability, with cells proliferating exponentially under favorable conditions and entering a stationary phase when resources become scarce. This transition is mediated by a regulatory mechanism known as the stringent response, which allows bacteria to adapt to nutrient deprivation by modulating gene expression and metabolic activity.During nutrient scarcity, intracellular amino acid levels decline. It results in the accumulation of uncharged tRNAs...
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Aminoglycosides constitute a highly potent class of bactericidal antibiotics that exert their antimicrobial effects by targeting the bacterial ribosome, specifically disrupting protein synthesis. These polycationic molecules consist of amino-modified sugars linked via glycosidic bonds to an aminocyclitol core such as 2-deoxystreptamine or streptamine. Their strong positive charges facilitate tight binding to the negatively charged phosphate backbone of ribosomal RNA (rRNA), primarily at the 16S...
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Related Experiment Video

Updated: Apr 22, 2026

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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Harnessing toxin-mediated ribosome stalling as a complementary tool to annotate bacterial ORFs.

Eduardo A Troian1, Valdir C Barth1,2, Unnati Chauhan1

  • 1Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ 08854, United States.

Nucleic Acids Research
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Summary

Mycobacterium tuberculosis toxin VapC4 causes ribosome stalling, revealing new Cys-containing open reading frames (ORFs). This novel method, using ribosome stalling, efficiently detects and validates previously unannotated ORFs in Mtb genomes.

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Area of Science:

  • Microbiology
  • Genomics
  • Molecular Biology

Background:

  • The Mycobacterium tuberculosis (Mtb) VapC4 toxin targets and inactivates tRNACys, causing ribosome stalling at cysteine codons.
  • Existing methods for identifying novel open reading frames (ORFs) can be limited.

Purpose of the Study:

  • To develop and validate a novel genome annotation tool using toxin-induced ribosome stalling.
  • To identify and characterize previously unannotated cysteine-containing ORFs in Mtb.

Main Methods:

  • Utilized 5' RNA-seq to map ribosome positions stalled by VapC4 toxin.
  • Employed proteogenomics and mass spectrometry for ORF validation.
  • Compared results with previously published Ribo-RET data.

Main Results:

  • Successfully mapped stalled ribosomes to identify new Cys-containing Mtb ORFs without requiring Ribo-seq.
  • Unmasked 96 unannotated ORFs, with 54% being small ORFs (≤50 amino acids).
  • Validated 69% of the identified ORFs via mass spectrometry, including four matched to synthetic controls.

Conclusions:

  • Toxin-mediated ribosome stalling is a robust and innovative genome annotation tool for mycobacteria and other bacteria.
  • This method complements existing genome annotation techniques and offers unique advantages.
  • Identified ORFs provide functional insights, including potential roles in Cys-responsiveness and encoding novel proteins.