Download ROEE AMIT DEPARTMENT OF BIOTECHNOLOGY AND FOOD

SYNTHETIC BIOLOGY
GAMES WITH RNA IN
BACTERIA
ROEE AMIT
DEPARTMENT OF BIOTECHNOLOGY AND FOOD ENGINEERING
TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY
7TH ANNUAL WINTER Q-BIO MEETING IN HAWAII
FEB. 24TH, 2017
WHY RNA? HOW IS IT DIFFERENT FROM DNA?
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RNA makes secondary and
tertiary structure.
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RNA degrades.
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RNA-based or translational
regulation is not well
understood.
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RNA regulation is faster than
transcriptional regulation.
WHY SYNTHETIC BIOLOGY?
SYSTEMATIC LARGE SCALE VARIATIONS OF DIFFERENT
REGULATORY ARCHITECTURES.
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RBP-RNA regulation
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RNA-based insulation in bacteria
SYNTHETIC BIOLOGY FORCES US TO TEST WHAT WE THINK
WE UNDERSTAND ABOUT BIOLOGY!
Lior Levy
Leon Anavy
Noa Katz
SYNTHETIC RBP-BASED REGULATION
RNA VERSION OF BUILDING SYNTHETIC PROMOTERS
IN VIVO RBP BINDING ASSAY
CAN CHARACTERIZE RBP BINDING AFFINITIES
CURRENTLY EXPANDING TO A LARGE SCALE SCREEN.
BASAL LEVEL PERIODICITY - WHY?
RIBOSOME CAN UNWIND HAIRPIN STRUCTURES
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Rate of unwinding is position dependent with 3nt periodicity.
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Every binding site tested exhibited this behavior.
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Kinetic hopping model can explain this behavior.
SYNTHETIC TRANSLATION REPRESSION
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RBPs can repress translation only when the binding site is encoded within the
initiation region.
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Mechanism is likely due to blocking of 70S formation
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No evidence for scanning 70S initiation.
SUMMARY
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Showed that Ribosome can unwind hairpin of various sizes inside
CDS.
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Hairpins encoded within initiation region do not affect translation.
This goes against current understanding.
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RBP can repress expression in the initiation region or 5’UTR (data
not shown).
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Multiple RBP binding sites alter binding affinity - stabilize one
another (data not shown).
WHAT IS CONTEXT?
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Sequence level effects that may be embedded in the core
“element”.
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Secondary regulatory functions that only emerge when the
elements is taken out of “context”.
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Encoded in sparse or not easily identifiable sequence elements.
EXAMPLE: BACTERIAL INSULATION
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We engineered this synthetic configuration and found silencing - how
can one promoter silence another?
IT’S NOT IN THE DNA….
LOOKS LIKE IT’S IN THE RNA
SO HOW IS IT ENCODED?
USE A SYNTHETIC LIBRARY APPROACH…
135 MUTATIONS OF GLNKP IN PARALLEL
THERE IS IT? - IN THE BLUE REGION!
WHAT ABOUT OTHER SIGMA54
PROMOTERS?
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3-5 nt PSSM. Impossible to identify by looking at a single or handful
of variants!
HOW IS INSULATION ENCODED?
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Strength of insulation is proportional to how many CT-rich motifs are
encoded within the core region, and how close they are to RBS.
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Phenomenon is enriched for σ54 promoter sub-class in E.coli
WHAT ABOUT SYNTHETIC BIOLOGY?
NEW DESIGN RULE FOR CIRCUIT DESIGN
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Model that takes into account the new insulation phenomenon
outperforms RBS calculators.
SUMMARY
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Synthetic OLs can help unravel “context” dependent effects in vivo.
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Bacterial insulators predominantly encoded in sig54 promotes.
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Insulators are encode by multiple sparse 3-5 nt CT-(purine-rich)
motif.
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Mechanism is anti-Shine Dalgarno:Shine Dalgarno binding.
ACKNOWLEDGEMENTS
Insulators
RBP-RNA Interactions
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Lior Levy
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Noa Katz
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Leon Anavy
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Ronni Cohen
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Oz Solomon
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Beate Kauffman
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Roni Cohen
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Sarah Goldberg
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Orna Atar
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Shilo Ohayon
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Sarah Goldberg
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Orna Atar
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Zohar Yakhini
Positions Available:
Ph.D Students
Post Docs