Folding of riboswitches during RNA transcription
•Background information
•What are riboswitches?
•Folding of riboswitch terminatorsEfficient folding of hairpins
Evolution of terminator sequences
withBen Sauerwine (Carnegie Mellon, Physics)
“Central Dogma” of Molecular Biology
DNA
RNA
5´ untranslated untranslated 3´Coding Region
Transcription
Translation
Transcriptionstart
Transcriptionstop
... ...
Protein
(RNA World hypothesis)
messenger RNA structure
Primary structure (sequence):UUACAAUAUAAUAGGAACACUCAUAUAAUCGCGUGGAUAUGGCACGCAAGUUUCUACCGGGCACCGUAAAUGUCCGACUAUGGGUGAGCAAUGGAACCGCACGUGUACGGUUUUUUGUGAUAUCAGCAUUGCUUGCUCUUUAUUUGAGCGGGCAAUGCUUUUUUUAUUCUCAUAACGGAGGUAGACAGGAUGGAAGCACUGAAACGGAAAAUAGAGGAAGAAGGCGUCGUAUUAUCUGAUCAGGUAUUGAAAGUGGAUUCUUUUUUGAAUCACCAAAUUGAUCCGCUGCUUAUGCAGAGAAUUGGUGAUGAAUUUGCGUCUAGGUUUGCAAAAGACGGUAUUACCAAAAUUGUGACAAUCGAAUCAUCAGGUAUCGCUCCCGCUGUAAUGACGGGCUUGAAGCUGGGUGUGCCAGUUGUCUUCGCGAGAAAGCAUAAAUCGUUAACACUCACCGACAACUUGCUGACAGCGUCUGUUUAUUCCUUUACGAAGCAAACAGAAAGCCAAAUCGCAGUGUCUGGGACCCACCUGUCGGAUCAGGAUCAUGUGCUGAUUAUCGAUGAUUUUUUGGCAAAUGGACAGGCAGCGCACGGGCUUGUGUCGAUUGUGAAGCAAGCGGGAGCUUCUAUUGCGGGAAUCGGCAUUGUUAUUGAAAAGUCAUUUCAGCCGGGAAGAGAUGAACUUGUAAAACUGGGCUACCGAGUGGAAUCUUUGGCAAGAAUUCAGUCUUUAGAAGAAGGAAAAGUGUCCUUCGUACAGGAGGUUCAUUCAUGA
(Bacillus Subtilis XPT gene)
• Folded secondary structure
• Complementary base pairing
• Can this really matter ?!?
Representations of Secondary Structure
....((((((((.....(((((.......)))))..........((((((.......))))))..))))))))....
Tertiary Structure(XPT “Aptamer”)
AUC=G(GU)
3´5´
5´ 3´
Riboswitch folding during transcription
Terminatorstem
Aptamerstem
Guanine stabilizes aptamerTerminator stalls transcription“XPT switched off”
G Guanine absentAntiterminator prevents termination“XPT switched on”
GG
Anti-terminator
“Gene Regulation by Riboswitches”Mandal & Breaker Nature Reviews (2004)
5´5´
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)
Sequence length 43 nt Transcription rate 50 nt/second
XPT Terminator Folding Success Rate
RealSequenceG=23.0 kCal/mol
Typical ShuffledSequenceG=23.8 kCal/mol
Atypical ShuffledSequenceG=25.0 kCal/mol
Real and Shuffled Sequences
MetastableG=7.9 kCal/mol
StableG=25.0 kCal/mol
MetastableG=3.4 kCal/mol
Atypical shuffled sequence - Alternate folds
Terminator Folding Success Rates
Bacillus Subtilis whole genome
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 (?)
Conclusions
• RNA secondary structure does matterSelf-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