Download - Riboswitches Sharon Epstein 30/03/2006 Frontiers in Metabolome sciences Feinberg Graduate School
Riboswitches
Sharon Epstein30/03/2006
Frontiers in Metabolome sciencesFeinberg Graduate School
Outline Introduction Concepts Evolution Structure Mechanisms Methods Examples Applications Conclusion
Introduction
Pubmed search for “riboswitches”: Reviews: 12 Articles: 57 Pubmed search for “micro RNA”: Reviews: 167 Articles: 740
Introduction
Riboswitches were “discovered” in the beginning of the 21st century
The idea was known but could not be proved
Introduction
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)
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
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 5’UTR
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
Evolution
Major difference in eukaryotes: localization
Present in introns and 3’UTR, not well studied and not much data available
Found in Arabidopsis and rice, on different splice variants (one regulated one not)
Structure
Riboswiches are composed of two interdependent but distinct domains:
Aptamer domain (responsible for binding of ligand)
Expression plataform (responsible for impacting gene expression)
Structure
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
Formation of intrinsic terminator stem
Inhibition of translation initiation
Self-cleaving mechanism
Methods
In line probing:
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
Examples
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
Coenzyme B12
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
Flavin Mononucleotide
Present upstream of genes for riboflavin biosynthesis and transport pathway
Binds FMN 100 folds more tightly then riboflavin (difference: one phosphate)
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%
S-adenosylmethionine
S-box motif – present mainly in gram-positive bacteria
Upstream of sulfur, cysteine, SAM and methionine pathways
1:1 stoichiomestry, dependent on Mg+2
S-adenosylmethionine
Lysine
Descriminates between l and d lysine
AEC (toxic analog) is also bound by riboswitch and resistant bacteria carry mutations
Potential drug target?
Glucosamine-6-phosphate
RNA undergoes rapid self cleavage upon binding of metabolite
Mutations that affect ribozyme activity de-repress the gene
Glycine
Two aptamer upstream of glycine cleaving proteins – each binds one glycine but increases affinity of the other aptamer – increase in sensitivity
Evolutionary advantage?
Glycine
Applications
Drug target (antimicrobial)
Molecular engineering
Conclusion
Evolutionary clues Non coding regions of RNA as
undiscovered regulatory domains New role of bioinformatics Possibilities
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)