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

....((((((((.....(((((.......)))))..........((((((.......))))))..))))))))....

TertiaryStructure

AU CG(GU)

Riboswitch folding during transcription

Terminatorstem

Aptamerstem

Guanine in solution, stabilizes aptamerTerminator forms, stalling polymeraseTranscription halts, Guanine not synthesized“XPT switched off”

G Guanine absentAntiterminator formsTranscription proceeds, Gaunine synthesized“XPT switched on”

GG

Anti-terminator

“Gene Regulation by Riboswitches”Mandal & Breaker Nature Reviews (2004)

Work in progress

1. Antiterminator lifetimeFirst passage time for escape from

metastable state(Ben Sauerwine, short talk)

2. Natural selection of terminator sequencesEvidence that terminators are selected for reliable folding(This talk)

Sequence length 43 ntTranscription rate 50 nt/second

RealSequence

Typical ShuffledSequence

Atypical ShuffledSequence

MetastableG=-7.9 kCal/mol

StableG=-25.0 kCal/mol

MetastableG=-3.4 kCal/mol

Atypical shuffled sequenceAlternate folds

Conclusions

RNA secondary structure does matter (sometimes)

Operation of riboswitch successfully modeled with Kinfold

Evidence for natural selection of efficiently folding sequences