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