Transcript Document

RNA-metabolite interactions
(riboswitches)
RNA aptamers
•
RNA aptamers are structures
that bind specifically to target
ligands
•
Many aptamers have been
generated in the laboratory
for a wide range of target
molecules (e.g. theophylline
aptamer)
•
Structural studies of aptamer-ligand complexes have
provided a wealth of information regarding RNA structure
and ligand interaction
•
Most aptamers exhibit conformational changes upon
binding to ligand (induced fit binding)
Natural aptamers are the basis of
riboswitches
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Natural aptamers reside in the non-coding segments of
messenger RNAs (mainly prokaryotic)
•
Interact directly (without the need for protein) with
metabolites to control gene expression
•
Typically provide a feedback mechanism for controlling
the expression of metabolic genes
metabolic product of a pathway inhibits expression of
proteins required to produce the metabolite
•
Most riboswitches are comprised of the aptamer and an
‘expression platform’ (some way of altering gene
expression)
Conserved RNA sequence involved in riboflavin synthesis
Flavin mononucleotide
RFN aptamer
Gelfand, M.S., Trends in Genetics (1999) 15: 439-442.
Genetic Control Mechanisms
Tom Cech, Nature (2004) 428: 263-264.
Genetic Control Mechanisms - RIBOSWITCHES
Tom Cech, Nature (2004) 428: 263-264.
Natural aptamers are the basis of
riboswitches
Riboswitches
•
RNA domains that modulate gene expression in response to
metabolite binding
Pre-Queuosine-I
2’-deoxyguanosine
Winkler & Breaker (2005) Annu Rev Microbiol 59: 487-517
Roth, et al. (2007) Nat Struct Mol Biol. 14: 308-317
Kim et al. (2007) Proc. Natl. Acad. Sci. 104: 16092-16097
Known riboswitch aptamers
Known riboswitch aptamers
Known riboswitch aptamers
Glycine riboswitch use of cooperativity
Known riboswitch aptamers
Glucosamine-6-phosphate riboswitch riboswitch is a ribozyme
Tom Cech, Nature (2004) 428: 263-264.
Riboswitches: RNA-mediated genetic control
Mandal et al. (2003)
Cell 113: 577-586
~4%
Winkler & Breaker (2005) Annu. Rev. Microbiol. 59: 487-517
Conformation change drives
riboswitch function
•
Conformation changes upon metabolite-binding alter
RNA structure and affect gene expression
•
Three known mechanism of action:
Transcription termination/antitermination
Translation control
RNA processing
Riboswitch mechanisms
•
Transcription termination
and anti-termination
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Translation initiation: RBS
accessibility
•
RNA processing:
Splicing or degradation
Winkler & Breaker (2005) Annu Rev
Microbiol 59:487-517.
Riboswitch mechanisms
Barrick & Breaker (2006) Scientific American 296: 50-57
TPP Riboswitch
•
Recognizes ligand
phosphate through metal
ion-mediated backbone
and nucleobase contacts
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Riboswitch regulation of
gene expression is
perturbed by the antibiotic
PTPP
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Riboswitch agonists or
antagonists: novel
antibiotics?
Sudarsan et al. (2005) Chem Biol 12:1325-1335
Serganov et al. (2006) Nature 441:1167-1171
Thore et al. (2006) Science 312:1208-1211
Thiamine pyrophosphate (TPP)
riboswitch
TPP-dependent modulation of spontaneous RNA cleavage
In-line attack technique
In-line attack technique
RNA is a high-affinity metabolite receptor
Apparent KD based on half maximal
modulation of RNA
Equilibrium dialysis technique
3H-thiamine
3H-thiamine
High sensitivity and selectivity of mRNA for
metabolite binding
Equilibrium dialysis
M3 = non-TPP binding mutant
Mutational analysis of the structure & function of thiM riboswitch
-gal reporter gene + riboswitch - control of gene expression?
Proposed mechanism for TPP-dependent deactivation of thiM translation
Additional Riboswitch Info
Tandem riboswitches exhibit complex gene control
Diverse tandem riboswitch configurations & functions
Orphan riboswitches
Crystal structure - guanine riboswitch
Crystal structure - guanine riboswitch
Guanine would have
an amino group here.
Crystal structure - TPP riboswitch
Crystal structure - SAM riboswitch
Crystal structure - GlcN6P riboswitch
Crystal structure – Lysine riboswitch