1. Riboswitch Regulation of Gene Expression
Created by Dr. Gail Mitchell Emilsson
In the laboratory of Dr. Ronald R. Breaker
at Yale University
2004

2. Riboswitches are present in untranslated regions of mRNAs
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.

4. Part I:
Gene Regulation by a
Typical Riboswitch

6. Bacterial riboswitches are present in the
5´ untranslated region of mRNAs.
Transcription is regulated by the gene promoter and transcription initiation factors …

7. RNA polymerase initiates transcription.
A long untranslated leader is produced first.
The RNA folds intramolecularly in local regions of complementarity, presumably, while transcription is proceeding.
Nascent RNA
RNA
polymerase
DNA template

8. Case 1:
Cellular concentration of metabolite is too low to occupy the riboswitch binding site.
Transcription and … 3 4 2 1
RNA
polymerase
RNA
polymerase

9. Transcription and intramolecular RNA folding continue.
Case 1:
Cellular concentration of metabolite is too low to occupy the riboswitch binding site.
Transcription and intramolecular RNA folding continue. 3 4 2 1 3 4 2 1
RNA
polymerase U A G

10. Transcription and intramolecular RNA folding continue.
Case 1:
Cellular concentration of metabolite is too low to occupy the riboswitch binding site.
Transcription and intramolecular RNA folding continue.
Translation is initiated. 3 4 2 1 U A G
Typically the new mRNA codes for a biosynthetic or transport protein that raises the intracellular level of the metabolite.
Ribosome
Gene regulation (next case) is accomplished by variations in the interactions of the regions highlighted in orange.

12. X X X X X X Case 2: Cellular concentration of metabolite (X) is high.
RNA polymerase produces the long untranslated leader region.
Intramolecular folding can lead to an alternate conformation. X X X X
Nascent RNA X X
RNA
polymerase
DNA template
The alternate riboswitch conformation is stable when metabolite is bound.

13. X X X X X X Case 2: Cellular concentration of metabolite (X) is high.
RNA polymerase produces the long untranslated leader region.
Transcription continues.
Intramolecular folding can lead to an alternate conformation. X X X X X X 3 4 2 1
RNA
polymerase U
The alternate riboswitch conformation is stable when metabolite is bound.

14. Case 2:
Cellular concentration of metabolite (X) is high.
Transcription continues.
Now, RNA folding leads to formation of an intrinsic terminator. X X X X X X X 3 4 2 1 3 4 2 1
RNA
polymerase U

15. X X X X X X Case 2: Cellular concentration of metabolite (X) is high.
Transcription continues.
Now, RNA folding leads to formation of an intrinsic terminator. U X X X X X X 3 4 2 1
RNA
polymerase
The transcript is never completed and the metabolite biosynthetic or transport protein is not produced.

17. Review X X X X X Case 1: Metabolite is limited. Case 2:
Metabolite is abundant. X U X X U A G
ORF 2 3 1 4 X X 3 4 2 1
Transcription is completed.
Transcription is terminated.
Proteins are downregulated.
Biosynthetic and/or transport proteins are expressed.

19. The Expanding Universe
Part II:
The Expanding Universe
of Riboswitches

21. Riboswitch Functions
Riboswitches were defined earlier by two main criteria:
These two activities are accomplished by two functionally separate domains on the RNA:
Direct (protein-free) binding of metabolite to RNA
Metabolite-dependent regulation of genes
A metabolite-binding
‘aptamer’ domain
and …
… an ‘expression platform’ for gene regulation. X 1 2 3 4 U U U U U

22. Riboswitch Functions
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 …
A metabolite-binding
‘aptamer’ domain
and …
… an ‘expression platform’ for gene regulation. X 1 2 3 4 U U U U U

23. Riboswitch Mechanisms
This movie showed the most common case for bacterial riboswitches: U X 5´
ORF
Ligand binding leads to
transcription termination and reduced gene expression. X

24. Riboswitch Mechanisms
Riboswitches also regulate translation and anti-termination …
Ligand binding leads to
transcription termination and reduced gene expression. X X 5´ U U U U U
ORF X 5´ A U G
ORF
Ligand binding sequesters
the Shine-Dalgarno sequence and reduces gene expression. X
AGGAGG U X 5´ A G
ORF
Ligand binding leads to
antiterminator formation and increased gene expression. X

25. Riboswitch Mechanisms
They also affect RNA integrity, and perhaps splicing and stability. X 5´
Ligand binding leads to
mRNA cleavage by a new natural ribozyme. X A U G
ORF X 5´ A U G
ORF AG
GUACGG
Ligand binding could control
splicing in eukayotes. A X 5´ U G
ORF
Possibly, ligand binding to the 3´ untranslated region could affect mRNA stability.

26. Riboswitch Ligands
There are 8 confirmed riboswitches with unique metabolite ligands.
Many more conserved RNA motifs are currently under investigation.
Figure shows the chemical structures of riboswitch ligands and schematics of conserved secondary structure in riboswitches.

27. Riboswitch Gene Regulation
Riboswitches 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.

28. Metabolite Recognition
Riboswitches are an economical way to see small molecules
Average metabolite
Coenzyme
B12
tRNA
Riboswitch
Relative sizes of some molecules
recognized for gene regulation
(metabolites and tRNAs) and some agents that
recognize them (riboswitches and TRAP complex).
11-mer TRAP complex

30. The End

31. Special thanks to: Sponsors of riboswitch research in
the Breaker laboratory
David and Lucile Packard Foundation
National Institutes of Health
National Science Foundation
For technical and
creative assistance
J. Kenneth Wickiser
For helpful comments
and suggestions
All members of the
Breaker laboratory

32. 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

33. For citations and additional information
For more information:
For citations and additional information