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![Page 1: Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004.](https://reader030.fdocuments.in/reader030/viewer/2022032802/56649e015503460f94aeb220/html5/thumbnails/1.jpg)
Riboswitch Regulation ofGene Expression
Created by Dr. Gail Mitchell Emilsson
In the laboratory of Dr. Ronald R. Breakerat Yale University
2004
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Riboswitches are present in untranslated regions of mRNAs. Their purpose is to regulate gene expression in response to binding small molecule metabolites.
Riboswitches are defined by two main criteria:
• Direct (protein-free) binding of metabolite to RNA• Metabolite-dependent regulation of genes
This movie demonstrates the molecular events common to most bacterial riboswitches. Resources are listed at the end of the movie.
![Page 3: Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004.](https://reader030.fdocuments.in/reader030/viewer/2022032802/56649e015503460f94aeb220/html5/thumbnails/3.jpg)
![Page 4: Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004.](https://reader030.fdocuments.in/reader030/viewer/2022032802/56649e015503460f94aeb220/html5/thumbnails/4.jpg)
Part I:
Gene Regulation by aTypical Riboswitch
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Bacterial riboswitches are present in the 5´ untranslated region of mRNAs.
Transcription is regulated by the gene promoter and transcription initiation factors …
![Page 7: Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004.](https://reader030.fdocuments.in/reader030/viewer/2022032802/56649e015503460f94aeb220/html5/thumbnails/7.jpg)
RNA polymerase initiates transcription.
The RNA folds intramolecularly in local regions of complementarity, presumably, while transcription is proceeding.
A long untranslated leader is produced first.
Nascent RNA
RNApolymerase
DNA template
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RNApolymerase
Case 1:Cellular concentration of metabolite is too low to occupy the riboswitch binding site.
Transcription and …
3 421 RNApolymerase
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UUUUU AUGRNA
polymerase
Case 1:Cellular concentration of metabolite is too low to occupy the riboswitch binding site.
Transcription and intramolecular RNA folding continue.
34
21 3 421
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UUUUU AUG
Case 1:Cellular concentration of metabolite is too low to occupy the riboswitch binding site.
Translation is initiated.
Ribosome
Typically the new mRNA codes for a biosynthetic or transport protein that raises the intracellular level of the metabolite.
Gene regulation (next case) is accomplished by variations in the interactions of the regions highlighted in orange.
Transcription and intramolecular RNA folding continue.
34
21
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Case 2:Cellular concentration of metabolite (X) is high.
Intramolecular folding can lead to an alternate conformation.RNA polymerase produces the long untranslated leader region.
The alternate riboswitch conformation is stable when metabolite is bound.
X X
X
X
X
RNApolymerase
X
Nascent RNA
DNA template
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Case 2:Cellular concentration of metabolite (X) is high.
Intramolecular folding can lead to an alternate conformation.RNA polymerase produces the long untranslated leader region.
The alternate riboswitch conformation is stable when metabolite is bound.
X
X X
X
X
X
Transcription continues.
UUUUURNA
polymerase
3 421
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Case 2:Cellular concentration of metabolite (X) is high.
X X
X
X
X
Transcription continues.
RNApolymerase
Now, RNA folding leads to formation of an intrinsic terminator.
UUUUU
X X
3 421 3 421
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Case 2:Cellular concentration of metabolite (X) is high.
X X
X
X
X
Transcription continues.
RNApolymerase
Now, RNA folding leads to formation of an intrinsic terminator.
UUUUU
X
The transcript is never completed and the metabolite biosynthetic or transport protein is not produced.
3 421
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ReviewCase 1:Metabolite is limited.
Case 2:Metabolite is abundant.
UUUUU
XUUUUU
AUG ORF
Transcription is completed.Biosynthetic and/or transport proteins are expressed.
Transcription is terminated.Proteins are downregulated.
X X
XX 3 421
3
4
21
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Part II:
The Expanding Universeof Riboswitches
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… an ‘expression platform’ for gene regulation.
A metabolite-binding‘aptamer’ domain and …
Riboswitch Functions
UUUUU
X
3 421
Riboswitches were defined earlier by two main criteria:
• Direct (protein-free) binding of metabolite to RNA
• Metabolite-dependent regulation of genes
These two activities are accomplished by two functionally separate domains on the RNA:
![Page 22: Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004.](https://reader030.fdocuments.in/reader030/viewer/2022032802/56649e015503460f94aeb220/html5/thumbnails/22.jpg)
… an ‘expression platform’ for gene regulation.
A metabolite-binding‘aptamer’ domain and …
Riboswitch Functions
UUUUU
X
3 421
• Direct (protein-free) binding of metabolite to RNA
• Metabolite-dependent regulation of genes
These two activities are accomplished by two functionally separate domains on the RNA:
Because of the modular nature of RNA structures, different types of expression platform can be linked to the conserved aptamer domain.
This leads to variations in riboswitch mechanism …
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Riboswitch MechanismsThis movie showed the most common case for bacterial riboswitches:
Ligand binding leads totranscription termination and reduced gene expression.
UUUUU
X
5´ ORFX
![Page 24: Riboswitch Regulation of Gene Expression Created by Dr. Gail Mitchell Emilsson In the laboratory of Dr. Ronald R. Breaker at Yale University 2004.](https://reader030.fdocuments.in/reader030/viewer/2022032802/56649e015503460f94aeb220/html5/thumbnails/24.jpg)
Riboswitch MechanismsRiboswitches also regulate translation and anti-termination …
Ligand binding leads totranscription termination and reduced gene expression.
UUUUU
X
5´ ORF
Ligand binding sequestersthe Shine-Dalgarno sequence and reduces gene expression.
Ligand binding leads toantiterminator formation and increased gene expression.UUUUU
X
5´ AUG ORF
X
X
X
5´ AUG ORFAGGAGG X
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Riboswitch MechanismsThey also affect RNA integrity, and perhaps splicing and stability.
Ligand binding leads tomRNA cleavage by a new natural ribozyme.
X
5´
Ligand binding could controlsplicing in eukayotes.
Possibly, ligand binding to the 3´ untranslated region could affect mRNA stability.
AUG ORF
X
5´ AUG ORFAGGUACGG
AAAAA
X
5´ AUG
ORF
X
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Riboswitch Ligands
Figure shows the chemical structures of riboswitch ligands and schematics of conserved secondary structure in riboswitches.
There are 8 confirmed riboswitches with unique metabolite ligands.Many more conserved RNA motifs are currently under investigation.
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Riboswitch Gene RegulationRiboswitches are an important mechanism of gene regulation.
For example, nearly 2% of the genes of the model organism Bacillus subtilis appear to be controlled by riboswitches.
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Metabolite Recognition
tRNARiboswitch
11-mer TRAP complex
CoenzymeB12
Average metabolite
Relative sizes of some molecules recognized for gene regulation (metabolites and tRNAs) and some agents that recognize them (riboswitches and TRAP complex).
Riboswitches are an economical way to see small molecules
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The End
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Special thanks to:
Sponsors of riboswitch research in the Breaker laboratory
David and Lucile Packard FoundationNational Institutes of HealthNational Science Foundation
For technical and creative assistance
J. Kenneth Wickiser
For helpful commentsand suggestions
All members of theBreaker laboratory
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Riboswitch team:
Ron Breaker
Jeff Barrick Gail Emilsson Izabela Puskarz
Ben Boese Mark Lee Adam Roth
Keith Corbino Jinsoo Lim Narasimhan Sudarsan
Smadar Cohen-Chalamish
Maumita Mandal Ken Wickiser
Margaret Ebert Shingo Nakamura Wade Winkler
Ali Nahvi
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For more information:
http://www.yale.edu/breaker
For citations and additional information
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