Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction Stephen J. Goldfless, Brian A. Belmont, Alexandra.

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Presentation transcript:

Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction Stephen J. Goldfless, Brian A. Belmont, Alexandra M. de Paz, Jessica F. Liu and Jacquin Niles presented by Alfred Ramirez and Lauren Berry : February 29, 2012

Background: Aptamer Selection Previously screened aptamers for binding to TetR Secondary structure involves two conserved motifs Mutation of conserved sequences affects TetR binding

Background: Design Overview

Design Principles and Approach Screen a library of known TetR-aptamer interactions for those that regulate translation Modify the selected aptamer to maximize translation efficiency Validate the translation regulation Optimize for modularity and streamlining

Screen: Aptamer Selection

Modification: Aptamer Minimization Aptamers and exhibited desired translation regulation. Modified aptamer to minimize stability, creating aptamer and 5-1.2m2

Validation: Translation Repression

Validation: Episomal Inducible Gene Expression

Validation: TRP1 Integrated Inducible Gene Expression

Optimization: Expanding Regulatory Potential Goal: Expand the scope of regulatory behavior while maintaining the aptamer as a validated, defined component.

Optimization: Logic Inversion

Optimization: Reduction of Translation Impact Authors observed that aptamer had a significant impact in gene expression levels compared to no aptamer. Goal: Minimize impact of the maximum protein output while preserving the regulatory function of the aptamer.

Optimization: Reduction of Translation Impact

Optimization: Modularity Goal: Assess the modularity of the aptamer in the context of different 5'-UTR.

Optimization: Modularity

Optimization: Streamlining the Selection of Functional Interactions Goal: Define strategy to rapidly identify new functional aptamer variants

Optimization: Streamlining the Selection of Functional Interactions Ura3p allows growth in -uracil media and causes cell death in +5-FOA media

Optimization: Streamlining the Selection of Functional Interactions

Conclusions Apatmer used to regulate protein expression at the RNA level Optimization of aptamer can change max expression and repression levels System is modular: able to use with different 5'-UTRs

Significance of System Host cell independent Biologically robust Modular Successful in vivo Future Work Organisms with poorly understood transcriptional regulation Further regulation of circuits

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