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Related Concept Videos

Riboswitches01:56

Riboswitches

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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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Transcriptional Regulation: Riboswitches01:23

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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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Ribosome Profiling02:24

Ribosome Profiling

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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.
Applications of ribosome profiling
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Types of RNA01:23

Types of RNA

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Overview
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.
RNA...
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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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Ribozymes02:47

Ribozymes

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The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
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Artificial riboswitch selection: A FACS-based approach.

Zohaib Ghazi1, Casey C Fowler, Yingfu Li

  • 1Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|February 20, 2014
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Summary

This study presents a method using fluorescence-activated cell sorting (FACS) to engineer custom riboswitches. This technique efficiently screens large libraries for desired regulatory functions in synthetic biology.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Riboswitches are versatile genetic elements with significant potential in synthetic biology.
  • Developing custom riboswitches de novo or modifying existing ones is crucial for expanding their applications.
  • Efficient screening methods are needed to identify functional riboswitches from large sequence libraries.

Purpose of the Study:

  • To describe a method for engineering custom riboswitches using fluorescence-activated cell sorting (FACS).
  • To provide protocols for optimizing FACS conditions and identifying functional riboswitches.

Main Methods:

  • Utilizing fluorescence-activated cell sorting (FACS) for high-throughput screening of riboswitch libraries.
  • Developing and optimizing experimental setups and protocols for FACS selection.
  • Employing follow-up assays for the identification and characterization of selected riboswitches.

Main Results:

  • Demonstration of FACS as an efficient tool for selecting riboswitches with desired activities.
  • Establishment of protocols for testing and optimizing FACS conditions for riboswitch engineering.
  • Successful identification and characterization of novel riboswitches through the described method.

Conclusions:

  • FACS is a powerful technique for the rapid and efficient selection of custom riboswitches.
  • The described methodology facilitates the de novo design and modification of riboswitches for synthetic biology.
  • This approach enhances the utility of riboswitches in diverse biological applications.