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 Gelfand, M.S., Trends in Genetics (1999) 15: Flavin mononucleotideRFN aptamer

Genetic Control Mechanisms Tom Cech, Nature (2004) 428:

Genetic Control Mechanisms - RIBOSWITCHES Tom Cech, Nature (2004) 428:

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: Roth, et al. (2007) Nat Struct Mol Biol. 14: Kim et al. (2007) Proc. Natl. Acad. Sci. 104:

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:

Mandal et al. (2003) Cell 113: Riboswitches: RNA-mediated genetic control Winkler & Breaker (2005) Annu. Rev. Microbiol. 59: ~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:

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: Serganov et al. (2006) Nature 441: Thore et al. (2006) Science 312:

Thiamine pyrophosphate (TPP) riboswitch

TPP-dependent modulation of spontaneous RNA cleavage In-line attack technique

RNA is a high-affinity metabolite receptor Apparent K D based on half maximal modulation of RNA

Equilibrium dialysis technique 3 H-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

Guanine would have an amino group here. Guanine would have an amino group here.

Crystal structure - TPP riboswitch

Crystal structure - SAM riboswitch

Crystal structure - GlcN6P riboswitch

Crystal structure – Lysine riboswitch