Riboswitches

1
Riboswitches

• Sharon Epstein
• 30/03/2006
• Frontiers in Metabolome sciences
• Feinberg Graduate School

2
Outline

• Introduction
• Concepts
• Evolution
• Structure
• Mechanisms
• Methods
• Examples
• Applications
• Conclusion

3
Introduction

• Pubmed search for riboswitches
• Reviews 12
• Articles 57
• Pubmed search for micro RNA
• Reviews 167
• Articles 740

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Introduction

• Riboswitches were discovered in the beginning
  of the 21st century
• The idea was known but could not be proved

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Introduction
6
Concept

• Riboswitches method of controlling gene
  expression. ()a sequence of RNA that, through
  its secondary and tertiary structure, selectively
  binds a specific metabolite. (Templeton et al,
  2005)

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Concept

• When a metabolite is bound the secondary and
  tertiary structure of the RNA changes affecting
  transcription and translation in prokaryotes and
  possibly mRNA processing in eukaryotes

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Evolution

• RNA world, possible mode of regulation in the
  absence of proteins
• General conservation of metabolites so far known
  to be involved in binding
• Present and studied in prokaryotes with
  differences in bacterial groups
• Present in prokaryotes mainly on 5UTR

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Evolution

• Another hypothesis it is more recent then RNA
  world but is present in different bacterial
  groups because of lateral transfer and repetitive
  re-invention
• Focus prokaryote

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Evolution

• Major difference in eukaryotes localization
• Present in introns and 3UTR, not well studied
  and not much data available
• Found in Arabidopsis and rice, on different
  splice variants (one regulated one not)

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Structure

• Riboswiches are composed of two interdependent
  but distinct domains
• Aptamer domain (responsible for binding of
  ligand)
• Expression plataform (responsible for impacting
  gene expression)

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Structure
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Mechanism

• Three known mechanisms
• Formation of intrinsic terminator stem (inhibits
  transcription by inducing its termination)
• Formation of complex hiding translation
  initiation site
• Self-cleaving mechanism

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Formation of intrinsic terminator stem
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Inhibition of translation initiation
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Self-cleaving mechanism
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Methods

• In line probing

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Methods

• Equilibrium dialysis radio labeled metabolite
  unequal distribution
• RNase H probing DNA complementary strand
  conformational change no cleavage
• Fluorescence FMN quenched in contact with
  riboswitch

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Examples
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Coenzyme B12

• One of the first to be discovered
• Upstream of cobalamin synthesis, porphyrin and
  cobalt transport and glutamate fermentation
• One of the largest aptamers with many connecting
  points

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Coenzyme B12
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Thiamine pyrophosphate

• Most widespread (also found by sequence
  similarity in plants)
• Identification of the riboswitch lead to function
  characterization of genes involved in the pathway

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Flavin Mononucleotide

• Present upstream of genes for riboflavin
  biosynthesis and transport pathway
• Binds FMN 100 folds more tightly then riboflavin
  (difference one phosphate)

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Guanine and adenine

• Same aptamer binds both only one point mutation
  C to U (forms base-pairing with ligand)
• Tertiary structure is similar sequence only 59

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S-adenosylmethionine

• S-box motif present mainly in gram-positive
  bacteria
• Upstream of sulfur, cysteine, SAM and methionine
  pathways
• 11 stoichiomestry, dependent on Mg2

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S-adenosylmethionine
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Lysine

• Descriminates between l and d lysine
• AEC (toxic analog) is also bound by riboswitch
  and resistant bacteria carry mutations
• Potential drug target?

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Glucosamine-6-phosphate

• RNA undergoes rapid self cleavage upon binding of
  metabolite
• Mutations that affect ribozyme activity
  de-repress the gene

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(No Transcript)
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Glycine

• Two aptamer upstream of glycine cleaving proteins
  each binds one glycine but increases affinity
  of the other aptamer increase in sensitivity
• Evolutionary advantage?

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Glycine
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Applications

• Drug target (antimicrobial)
• Molecular engineering

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Conclusion

• Evolutionary clues
• Non coding regions of RNA as undiscovered
  regulatory domains
• New role of bioinformatics
• Possibilities

40
Articles

• Winkler WC. (2005)
• Winkler WC et al. (2005)
• Tucker BJ et al. (2005)
• Templeton GW et al. (2004)
• Soukup JK et al. (2004)
• Mandal M et al. (2004)
• Nudler E et al. (2004)
• Vitreschak AG et al. (2004)
• Kaempfer R. (2003)
• Lai EC. (2003)
• Corbino KA et al. (2005)
• Altman S et al. (2005)