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A Systems Approach to Measuring the Binding Energy Landscapes of Transcription Factors. Sebastian J. Maerkl and Stephen R. Quake. Augusto Tentori October 10, 2008 20.309. Background. Systems Biology Understand Biological Networks - PowerPoint PPT Presentation
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A Systems Approach to Measuring the Binding Energy Landscapes of Transcription Factors
Sebastian J. Maerkl and Stephen R. Quake
Augusto Tentori
October 10, 2008
20.309
Background
Systems Biology Understand Biological Networks Genomic and proteomic methods, bottom-up genetic
network engineering
Need to quantitatively characterize unique-element interactions Many variables Transient interactions, low-affinity
Summary of Results
Developed high-throughput micro fluidic platform capable of detecting low-affinity transient binding events using mechanically-induced trapping of molecular interactions (MITOMI)
Mapped binding energy landscapes of four eukaryotic bHLH transcription factors Predicted in vivo function of two TF Tested base additivity assumption Tested whether basic region alone determines specificity of
bHLH TFs
Experiments
Binding energy landscapes of 4 bHLH TF MAX iso A MAX iso B Pho4p Cbf1p
bHLH TF generally bind to 5’-CANNTG-3’ E-box Mid-low nmolar affinities and koff’s of10^-2s^-1
Results
MAX iso A
67nM
MAX iso B
73.1nM
Pho4p
11.1nM
Cbf1p
16.6nM
Optimal binding sequence = CACGTG
ResultsMAX BMAX A
Tested additivity assumption Energetic and informatic role of a base in a given motif is
independent of its neighbors
Predicted only 44% of sequences below 2.5kcal/mol
Results Pho4p and Cbf1p show distinct functions yet have similar recognition
sequences CACGTGsG s=G,C and rTCACGTG r=A,G Measured possible permutations of 3 flanking base pairs to determine
how far base specific recognition extends.
Results Tested probability
of occupancy for regulatory sequence in 5814 yeast genes
Broad agreement with literature
Final Thoughts
Importance of high-throughput methods
Possible limitations of MITOMI
Thank you
Questions?
Fig. S4. (A) Overview of the experimental approach starting with a plain epoxy substrate tobe spotted with 2400 spots of a target DNA library. The finished microarray is then aligned and bonded to one of our microfluidic devices after which the surface is prepared, protein synthesized and MITOMI performed. (B) Micrograph of one of the microfluidic unit cells, shownhere again for reference. The dashed lines show which regions of the unit cell are schematically depicted in panels C-L. (C) Before any fluid is introduced into the device the chamber valve (green channel in panel B) is closed to prevent flooding of the DNA chamber.
(D) Next biotinylated BSA is introduced into our device which covalently bonds to the epoxy functional groups, both activating (via the biotin moieties) and passivating (epoxy groups) the surface. (E) A solution of neutravidin is introduced forming a monolayer on top of the biotinylated BSA layer. (F) The ”button” membrane is closed to protect the detection area from passivation via biotinylated BSA which passivates all accessible surface area. (G) Any unbound biotinylated BSA is purged before the ”button” membrane is opened again allowing access to the neutravidin surface below to which a biotinylated penta-histidine antibody is attached, concluding the surface derivatization. (H) ITT programmed with linear expression template is introduced into the device and allowed to flood the DNA chamber causing the solvation of the stored target DNA. Transcription factor is being synthesized and is pulled down to the surface by penta-histidine antibody. (I) The synthesized transcription factors functionally interact with the solvated target DNA pulling it down to the surface as well. (J) After 60-90 min the ”button” membrane is closed again mechanically trapping any molecular interactions taking place on the surface allowing all solution phase molecules to be washed away without loss of surface bound material (K-L).
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