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Folding of riboswitches during RNA transcription

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. Transcription start. Transcription stop.

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Folding of riboswitches during RNA transcription

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

  3. messenger RNA structure Primary structure (sequence): UUACAAUAUAAUAGGAACACUCAUAUAAUCGCGUGGAUAUGGCACGCAAGUUUCUACCGGGCACCGUAAAUGUCCGACUAUGGGUGAGCAAUGGAACCGCACGUGUACGGUUUUUUGUGAUAUCAGCAUUGCUUGCUCUUUAUUUGAGCGGGCAAUGCUUUUUUUAUUCUCAUAACGGAGGUAGACAGGAUGGAAGCACUGAAACGGAAAAUAGAGGAAGAAGGCGUCGUAUUAUCUGAUCAGGUAUUGAAAGUGGAUUCUUUUUUGAAUCACCAAAUUGAUCCGCUGCUUAUGCAGAGAAUUGGUGAUGAAUUUGCGUCUAGGUUUGCAAAAGACGGUAUUACCAAAAUUGUGACAAUCGAAUCAUCAGGUAUCGCUCCCGCUGUAAUGACGGGCUUGAAGCUGGGUGUGCCAGUUGUCUUCGCGAGAAAGCAUAAAUCGUUAACACUCACCGACAACUUGCUGACAGCGUCUGUUUAUUCCUUUACGAAGCAAACAGAAAGCCAAAUCGCAGUGUCUGGGACCCACCUGUCGGAUCAGGAUCAUGUGCUGAUUAUCGAUGAUUUUUUGGCAAAUGGACAGGCAGCGCACGGGCUUGUGUCGAUUGUGAAGCAAGCGGGAGCUUCUAUUGCGGGAAUCGGCAUUGUUAUUGAAAAGUCAUUUCAGCCGGGAAGAGAUGAACUUGUAAAACUGGGCUACCGAGUGGAAUCUUUGGCAAGAAUUCAGUCUUUAGAAGAAGGAAAAGUGUCCUUCGUACAGGAGGUUCAUUCAUGA (Bacillus Subtilis XPT gene) • Folded secondary structure • Complementary base pairing • Can this really matter ?!?

  4. Representations of Secondary Structure AU C=G (GU) 3´ 5´ 5´ 3´ 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)

  6. 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)

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

  8. 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

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

  10. Terminator Folding Success Rates Bacillus Subtilis whole genome

  11. 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 (?)

  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

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