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Related Concept Videos

Transcriptional Regulation: Riboswitches01:23

Transcriptional Regulation: Riboswitches

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Riboswitches are RNA elements that regulate gene expression by altering their secondary structures in response to specific effector molecules. These elements, located in the leader regions of certain mRNAs, act as transcriptional regulators by toggling between alternative conformations to control downstream gene expression. Riboswitch-mediated regulation is a precise mechanism for modulating biosynthetic pathways, as exemplified by the riboflavin biosynthesis pathway in Bacillus...
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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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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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Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use.
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Translational Regulation01:29

Translational Regulation

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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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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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Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch.

Stefan Crielaard1, Rick Maassen1, Tess Vosman1

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Researchers developed photoaffinity probes to study flavin mononucleotide (FMN) riboswitches. This method quantifies ligand binding and maps binding sites, even in live bacteria, advancing antibiotic and sensor development.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • RNA Biology

Background:

  • Riboswitches are regulatory RNA molecules controlling gene expression.
  • Flavin mononucleotide (FMN) riboswitches are key targets for antibiotics and biosensors.
  • Understanding FMN riboswitch ligand binding and folding is crucial for their application.

Purpose of the Study:

  • To develop and apply affinity-based profiling for FMN riboswitch characterization.
  • To quantitatively measure ligand binding and map binding sites.
  • To investigate riboswitch folding and function in cellular contexts.

Main Methods:

  • Design and synthesis of photoreactive ligands for photoaffinity labeling.
  • Quantitative measurement of ligand binding using covalent interactions.
  • Analysis of riboswitch structural folding under varying conditions (temperature, cations).
  • Integration of photoaffinity labeling with reverse transcription for nucleotide-resolution mapping.
  • Application of the method to cellular extracts and live bacteria.

Main Results:

  • Selective photoaffinity labeling of the FMN riboswitch was achieved.
  • Quantitative binding data was obtained, demonstrated with roseoflavin.
  • Conditional riboswitch folding was measured as a function of environmental factors.
  • Ligand binding sites within the aptamer domain were mapped with single-nucleotide resolution.
  • Endogenous FMN riboswitch folding and antibiotic binding were demonstrated in *Bacillus subtilis*.

Conclusions:

  • Photoaffinity labeling provides a powerful tool for studying riboswitch-ligand interactions.
  • This method enables quantitative binding measurements and precise mapping of binding sites.
  • The approach is applicable to complex biological samples, including live bacteria, offering insights into riboswitch function in vivo.