1. Folding of riboswitches during RNA transcription Background information What are riboswitches? Folding of riboswitch terminators Efficient folding of hairpins Evolution of terminator sequences with Ben Sauerwine (Carnegie Mellon, Physics)

2. “Central Dogma” of Molecular Biology DNA RNA 5´ untranslated untranslated 3´ Coding Region TranscriptionTranslation Transcription start Transcription stop... Protein (RNA World hypothesis)

3. messenger RNA structure Primary structure (sequence): UUACAAUAUAAUAGGAACACUCAUAUAAUCGCGUGGAUAUGGCACGCAAGUUUCUACCGGGCACCG UAAAUGUCCGACUAUGGGUGAGCAAUGGAACCGCACGUGUACGGUUUUUUGUGAUAUCAGCAUUG CUUGCUCUUUAUUUGAGCGGGCAAUGCUUUUUUUAUUCUCAUAACGGAGGUAGACAGGAUGGAAG CACUGAAACGGAAAAUAGAGGAAGAAGGCGUCGUAUUAUCUGAUCAGGUAUUGAAAGUGGAUUCUU UUUUGAAUCACCAAAUUGAUCCGCUGCUUAUGCAGAGAAUUGGUGAUGAAUUUGCGUCUAGGUUU GCAAAAGACGGUAUUACCAAAAUUGUGACAAUCGAAUCAUCAGGUAUCGCUCCCGCUGUAAUGACG GGCUUGAAGCUGGGUGUGCCAGUUGUCUUCGCGAGAAAGCAUAAAUCGUUAACACUCACCGACAA CUUGCUGACAGCGUCUGUUUAUUCCUUUACGAAGCAAACAGAAAGCCAAAUCGCAGUGUCUGGGAC CCACCUGUCGGAUCAGGAUCAUGUGCUGAUUAUCGAUGAUUUUUUGGCAAAUGGACAGGCAGCGC ACGGGCUUGUGUCGAUUGUGAAGCAAGCGGGAGCUUCUAUUGCGGGAAUCGGCAUUGUUAUUGAA AAGUCAUUUCAGCCGGGAAGAGAUGAACUUGUAAAACUGGGCUACCGAGUGGAAUCUUUGGCAAG AAUUCAGUCUUUAGAAGAAGGAAAAGUGUCCUUCGUACAGGAGGUUCAUUCAUGA (Bacillus Subtilis XPT gene) Folded secondary structure Complementary base pairing Can this really matter ?!?

4. Representations of Secondary Structure....((((((((.....(((((.......)))))..........((((((.......))))))..)))))))).... Tertiary Structure (XPT “Aptamer”) AUC=G(GU)AUC=G(GU) 3´5´ 3´

5. Riboswitch folding during transcription Terminator stem Aptamer stem Guanine stabilizes aptamer Terminator stalls transcription “XPT switched off” G Guanine absent Antiterminator prevents termination “XPT switched on” Anti- terminator “Gene Regulation by Riboswitches” Mandal & Breaker Nature Reviews (2004) 5´

6. Work in progress 1.Antiterminator lifetime First passage time for escape from metastable state (Ben Sauerwine, short talk) 2.Natural selection of terminator sequences Evidence that terminators are selected for reliable folding (This talk) Both simulated by Kinfold (Vienna RNA)

7. Sequence length 43 nt Transcription rate 50 nt/second XPT Terminator Folding Success Rate

8. Real Sequence  G=  23.0 kCal/mol Typical Shuffled Sequence  G=  23.8 kCal/mol Atypical Shuffled Sequence  G=  25.0 kCal/mol Real and Shuffled Sequences

9. Metastable  G=  7.9 kCal/mol Stable  G=  25.0 kCal/mol Metastable  G=  3.4 kCal/mol Atypical shuffled sequence - Alternate folds

10. Terminator Folding Success Rates Bacillus Subtilis whole genome

11. How does it work? 1.Placement of specifically-binding nucleotides (e.g. C) promiscuously-binding nucleotides (e.g. G) 2. Poorly-folding sequences removed from population (?)

12. 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