rna-metabolite interactions (riboswitches). rna aptamers rna aptamers are structures that bind...
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RNA-metabolite interactions (riboswitches)
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RNA aptamers
• RNA aptamers are structuresthat bind specifically to targetligands
• Many aptamers have beengenerated in the laboratoryfor a wide range of targetmolecules (e.g. theophyllineaptamer)
• 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)
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Natural aptamers are the basis of riboswitches
• 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)
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Conserved RNA sequence involved in riboflavin synthesis
Gelfand, M.S., Trends in Genetics (1999) 15: 439-442.
Flavin mononucleotide RFN aptamer
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Genetic Control Mechanisms
Tom Cech, Nature (2004) 428: 263-264.
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Genetic Control Mechanisms - RIBOSWITCHES
Tom Cech, Nature (2004) 428: 263-264.
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Natural aptamers are the basis of riboswitches
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• RNA domains that modulate gene expression in response to metabolite binding
Riboswitches
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
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Known riboswitch aptamers
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Known riboswitch aptamers
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Known riboswitch aptamersGlycine riboswitch -use of cooperativity
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Known riboswitch aptamersGlucosamine-6-phosphate riboswitch -
riboswitch is a ribozyme
Tom Cech, Nature (2004) 428: 263-264.
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Mandal et al. (2003) Cell 113: 577-586
Riboswitches: RNA-mediated genetic control
Winkler & Breaker (2005) Annu. Rev. Microbiol. 59: 487-517
~4%
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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
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• Transcription termination and anti-termination
• Translation initiation: RBS accessibility
• RNA processing:Splicing or degradation
Riboswitch mechanisms
Winkler & Breaker (2005) Annu Rev Microbiol 59:487-517.
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Riboswitch mechanisms
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Barrick & Breaker (2006) Scientific American 296: 50-57
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• Recognizes ligand phosphate through metal ion-mediated backbone and nucleobase contacts
• Riboswitch regulation of gene expression is perturbed by the antibiotic PTPP
• Riboswitch agonists or antagonists: novel antibiotics?
TPP Riboswitch
Sudarsan et al. (2005) Chem Biol 12:1325-1335Serganov et al. (2006) Nature 441:1167-1171
Thore et al. (2006) Science 312:1208-1211
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Thiamine pyrophosphate (TPP)
riboswitch
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TPP-dependent modulation of spontaneous RNA cleavage
In-line attack technique
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In-line attack technique
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RNA is a high-affinity metabolite receptor
Apparent KD based on half maximal modulation of RNA
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Equilibrium dialysis technique
3H-thiamine 3H-thiamine
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High sensitivity and selectivity of mRNA for metabolite binding
Equilibrium dialysisM3 = non-TPP binding mutant
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Mutational analysis of the structure & function of thiM riboswitch-gal reporter gene + riboswitch - control of gene expression?
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Proposed mechanism for TPP-dependent deactivation of thiM translation
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Additional Riboswitch Info
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Tandem riboswitches exhibit complex gene control
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Diverse tandem riboswitch configurations & functions
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Orphan riboswitches
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Crystal structure - guanine riboswitch
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Crystal structure - guanine riboswitch
Guanine would have Guanine would have an amino group here.an amino group here.
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Crystal structure - TPP riboswitch
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Crystal structure - SAM riboswitch
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Crystal structure - GlcN6P riboswitch
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Crystal structure – Lysine riboswitch