Riboswitch Regulation ofGene Expression

Created by Dr. Gail Mitchell Emilsson

In the laboratory of Dr. Ronald R. Breakerat Yale University

2004

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.

Part I:

Gene Regulation by aTypical Riboswitch

Bacterial riboswitches are present in the 5´ untranslated region of mRNAs.

Transcription is regulated by the gene promoter and transcription initiation factors …

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

RNApolymerase

Case 1:Cellular concentration of metabolite is too low to occupy the riboswitch binding site.

Transcription and …

3 421 RNApolymerase

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

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

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

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

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

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

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

Part II:

The Expanding Universeof Riboswitches

… 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:

… 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 …

Riboswitch MechanismsThis movie showed the most common case for bacterial riboswitches:

Ligand binding leads totranscription termination and reduced gene expression.

UUUUU

X

5´ ORFX

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

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

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.

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.

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

The End

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

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

For more information:

http://www.yale.edu/breaker

For citations and additional information