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