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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 • 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 • 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 • Riboswitch regulation of gene expression is perturbed by the antibiotic PTPP • 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