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RNA-metabolite interactions (riboswitches)
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)
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)
Conserved RNA sequence involved in riboflavin synthesis
Gelfand, M.S., Trends in Genetics (1999) 15: 439-442.
Flavin mononucleotide RFN aptamer
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
• 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
Known riboswitch aptamers
Known riboswitch aptamers
Known riboswitch aptamersGlycine riboswitch -use of cooperativity
Known riboswitch aptamersGlucosamine-6-phosphate riboswitch -
riboswitch is a ribozyme
Tom Cech, Nature (2004) 428: 263-264.
Mandal et al. (2003) Cell 113: 577-586
Riboswitches: RNA-mediated genetic control
Winkler & Breaker (2005) Annu. Rev. Microbiol. 59: 487-517
~4%
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
• 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.
Riboswitch mechanisms
Barrick & Breaker (2006) Scientific American 296: 50-57
• 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
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 dialysisM3 = 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 Guanine would have an amino group here.an amino group here.
Crystal structure - TPP riboswitch
Crystal structure - SAM riboswitch
Crystal structure - GlcN6P riboswitch
Crystal structure – Lysine riboswitch