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Riboswitches

Riboswitches

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.
The aptamer has high specificity for a particular metabolite which allows riboswitches to specifically regulate...

Reporter Genes

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.

Ribosome Profiling

Ribosome Profiling

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.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...

Transcriptional Regulation: Riboswitches

Transcriptional Regulation: Riboswitches

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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High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

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Reporter Gene-Based Screening for TPP Riboswitch Activators.

Christina E Lünse^1,2, Günter Mayer³

¹Life and Medical Sciences Institute, University of Bonn, Gerhard-Domagk-Str. 1, Bonn, 53121, Germany. christina.luense@yale.edu.

Methods in Molecular Biology (Clifton, N.J.)

|November 23, 2016

Summary

Researchers developed a reporter gene assay to find new antibacterial drugs targeting essential bacterial pathways. This method identifies compounds activating the thiamine pyrophosphate (TPP) riboswitch, offering a novel strategy against drug-resistant pathogens.

Keywords:

Antibiotics Miller assay Thiamine pyrophosphate Translational fusion lacZ reporter gene

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Area of Science:

Microbiology
Molecular Biology
Drug Discovery

Background:

Increasing prevalence of multidrug-resistant bacteria and hospital-acquired infections necessitates novel antibacterial agents.
Riboswitches, RNA molecules regulating gene expression, are promising drug targets due to their role in essential metabolic pathways.
Thiamine pyrophosphate (TPP) riboswitches control vital metabolic processes in bacteria.

Purpose of the Study:

To develop and validate a reporter gene-based screening assay for identifying compounds that activate bacterial riboswitches.
To discover novel small molecules that modulate the thiamine pyrophosphate (TPP) riboswitch.
To establish a versatile platform for targeting essential metabolic pathways in bacteria.

Main Methods:

A reporter gene system was engineered to detect the activation of the thiamine pyrophosphate (TPP) riboswitch.
High-throughput screening was employed to identify compounds that induce riboswitch-mediated gene expression.
The assay's adaptability for other riboswitch classes was assessed.

Main Results:

A functional reporter gene assay was successfully established for screening TPP riboswitch activators.
The assay demonstrated sensitivity in identifying compounds that specifically target the TPP riboswitch.
The screening platform showed potential for broad applicability across different riboswitch classes.

Conclusions:

The developed reporter gene assay provides an effective method for discovering novel antibacterial compounds targeting riboswitches.
This approach offers a promising strategy for combating infections caused by multidrug-resistant bacteria.
The adaptable nature of the assay facilitates the exploration of diverse essential metabolic pathways for therapeutic intervention.