Folding of riboswitches during RNA transcription

1
Folding of riboswitches during RNA transcription
with Ben Sauerwine (Carnegie Mellon, Physics)

• Background information
• What are riboswitches?
• Folding of riboswitch terminators
• Efficient folding of hairpins
• Evolution of terminator sequences

2
Central Dogma of Molecular Biology
DNA
Coding Region
5 untranslated
untranslated 3
RNA
Protein
(RNA World hypothesis)
3
messenger RNA structure
Primary structure (sequence) UUACAAUAUAAUAGGAACAC
UCAUAUAAUCGCGUGGAUAUGGCACGCAAGUUUCUACCGGGCACCGUAAA
UGUCCGACUAUGGGUGAGCAAUGGAACCGCACGUGUACGGUUUUUUGUGA
UAUCAGCAUUGCUUGCUCUUUAUUUGAGCGGGCAAUGCUUUUUUUAUUCU
CAUAACGGAGGUAGACAGGAUGGAAGCACUGAAACGGAAAAUAGAGGAAG
AAGGCGUCGUAUUAUCUGAUCAGGUAUUGAAAGUGGAUUCUUUUUUGAAU
CACCAAAUUGAUCCGCUGCUUAUGCAGAGAAUUGGUGAUGAAUUUGCGUC
UAGGUUUGCAAAAGACGGUAUUACCAAAAUUGUGACAAUCGAAUCAUCAG
GUAUCGCUCCCGCUGUAAUGACGGGCUUGAAGCUGGGUGUGCCAGUUGUC
UUCGCGAGAAAGCAUAAAUCGUUAACACUCACCGACAACUUGCUGACAGC
GUCUGUUUAUUCCUUUACGAAGCAAACAGAAAGCCAAAUCGCAGUGUCUG
GGACCCACCUGUCGGAUCAGGAUCAUGUGCUGAUUAUCGAUGAUUUUUUG
GCAAAUGGACAGGCAGCGCACGGGCUUGUGUCGAUUGUGAAGCAAGCGGG
AGCUUCUAUUGCGGGAAUCGGCAUUGUUAUUGAAAAGUCAUUUCAGCCGG
GAAGAGAUGAACUUGUAAAACUGGGCUACCGAGUGGAAUCUUUGGCAAGA
AUUCAGUCUUUAGAAGAAGGAAAAGUGUCCUUCGUACAGGAGGUUCAUUC
AUGA (Bacillus Subtilis XPT gene)

• Folded secondary structure
• Complementary base pairing
• Can this really matter ?!?

4
Representations of Secondary Structure
A?U CG (G?U)
3
5
3
5
Tertiary Structure (XPT Aptamer)
....((((((((.....(((((.......)))))..........((((((
.......))))))..))))))))....
5
Riboswitch folding during transcription
Anti- terminator
Terminator stem
G
5
5
Aptamer stem
G
Guanine absent Antiterminator prevents
termination XPT switched on
Guanine stabilizes aptamer Terminator stalls
transcription XPT switched off
Gene Regulation by Riboswitches Mandal
Breaker Nature Reviews (2004)
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Work in progress

• Antiterminator lifetime
• First passage time for escape from metastable
  state (Ben Sauerwine, short
  talk)

• Natural selection of terminator sequences
• Evidence that terminators are selected for
  reliable folding (This talk)

Both simulated by Kinfold (Vienna RNA)
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XPT Terminator Folding Success Rate
Sequence length 43 nt Transcription rate 50
nt/second
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Real and Shuffled Sequences
Real Sequence ?G?23.0 kCal/mol
Typical Shuffled Sequence ?G?23.8 kCal/mol
Atypical Shuffled Sequence ?G?25.0 kCal/mol
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Atypical shuffled sequence - Alternate folds
Stable ?G?25.0 kCal/mol
Metastable ?G?3.4 kCal/mol
Metastable ?G?7.9 kCal/mol
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Terminator Folding Success Rates
Bacillus Subtilis whole genome
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How does it work?

• Placement of specifically-binding nucleotides
  (e.g. C) promiscuously-binding nucleotides (e.g.
  G)

2. Poorly-folding sequences removed from
population (?)
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Conclusions

• RNA secondary structure does matter
• Self-regulation (riboswitches, microRNA, ...)
• Operation of riboswitch successfully modeled
  with Kinfold (Vienna RNA)
• Aptamer/Antiterminator/Terminator competition
• Evidence for natural selection of reliably
  folding sequences
• Probability of no selection P 0.01