Information Storage and Processing in Biological Systems: A seminar - - PowerPoint PPT Presentation
Information Storage and Processing in Biological Systems: A seminar course for the Natural Sciences Sept. 11 Biological Information, Sept 16 DNA, Gene regulation Sept 18 Translation and Proteins Sept 23 Enzymes and Signal Transduction
Biological Information, Sept 16 DNA, Gene regulation Sept 18 Translation and Proteins Sept 23 Enzymes and Signal Transduction Sept 25 Biochemical Networks Sept 30 Simple Genetic Networks (Dr. Jacob) Oct 2 Evolution, Evolvability and Robustness Oct 5 Adventures in multicellularity
(Britten-Davidson)
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(genetic regulatory networks)
within the set of genes
gene X gene Y
Promoter X Promoter Y
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(genetic regulatory networks)
within the set of genes
gene X gene Y
Promoter X Promoter Y
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(genetic regulatory networks)
within the set of genes
gene X gene Y
Promoter X
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(genetic regulatory networks)
gene X gene Y
By convention we simplify these diagrams as follows:
Promoter X
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(genetic regulatory networks)
Denotes positive regulation Denotes negative regulation
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A photomicrograph of three cells showing the flagella filaments. Each filament forms an extend helix several cell lengths long. The filament is attached to the cell surface through a flexible ‘universal joint’ called the hook. Each filament is rotated by a reversible rotary motor, the direction of the motor is regulated in response to changing environmental conditions.
Rotationally averaged reconstruction of electron micrographs of purified hook-basal
are the L ring, P ring, MS ring, and C ring. (Digital print courtesy of David DeRosier, Brandeis University.)
Checkpoint mechanism ensures that Class 3 genes are not transcribed before functional basal body-hook structures are completed.
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Positive transcriptional regulators Alternative sigma factors Anti-sigma factors Temporal regulation
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A D C B E
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flhCD fliA flgM fliL fliE fliF flgA flgB flhB n = 6 n = 6 fliD flgK fliC meche mocha flgM
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FlhCD
CRP,H-NS,OmpR
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FlhCD FliA FlgM Basal Body and Hook
CRP,H-NS,OmpR
inside
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FlhCD FliA FlgM Basal Body and Hook Filament Chemotaxis proteins Motor proteins
CRP,H-NS,OmpR
inside
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CRP - catabolite repression, carbohydrate metabolism OmpR - osmolarity IHF - growth state of cell? HdfR - ?
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inside
FlhDC expression leads to activation of Level 2 genes including the alternative sigma factor FliA and an anti sigma factor FlgM
Level 3 Genes FlgM accumulates in the cell and binds to FliA blocking its activity (i.e. interaction with RNA polymerase) preventing Level 3 gene expression.
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inside
Other level 2 genes required for Basal body and hook (BBH) assembly are made and begin to assemble in the membrane.
Level 3 Genes Basal Body and Hook Assembly
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inside
The Basal body and hook assembly are completed.
Level 3 Genes Completed Basal Body and Hook
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inside
The Basal body and hook assembly are completed.
Level 3 Genes Completed Basal Body and Hook FlgM is exported through the Basal Body and Hook Assembly
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inside
Level 3 gene expression is initiated.
Level 3 Genes Completed Basal Body and Hook FlgM is exported through the Basal Body and Hook Assembly.
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inside
Level 3 gene expression is initiated.
Level 3 Genes Completed Basal Body and Hook FliA can interact with RNA polymerase and activate Level 3 gene expression.
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inside
Filament
Level 3 gene products are added to the motility machinery including the (1) flagella filament, (2) motor proteins and (3) chemotaxis signal transduction system.
Motor proteins Chemotaxis proteins
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A D C B E
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flhCD fliA flgM fliL fliE fliF flgA flgB flhB n = 6 n = 6 fliD flgK fliC meche mocha flgM
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Small Defined Networks High Throughput / High Quality Expression Profiling Modeling, Simulation
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flhD flhC
flhDC promoter
Regulator
RNA polymerase
Organization of operon on chromosome.
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flhD flhC
flhDC promoter
Regulator
RNA polymerase
Organization of operon on chromosome. Reporter gene Clone a copy of the promoter into a reporter plasmid.
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flhD flhC Regulator
RNA polymerase
Reporter gene Both the flhDC genes and the reporter plasmid are regulated in the same way and thus the level of the reporter indicates the activity of the promoter.
Note that the strain still has a normal copy of the genes.
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Video microscopy
Multi-well plate reader
Automation: Both approaches are amenable to high throughput robotics
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Michael Elowitz, Rockefeller University
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Time [min] Fluorescence relative to max 0.01 0.1 0.6 C l a s s Operon 600
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Class 1 flhDC Class 2 fliL Class 2 fliE Class 2 fliF Class 2 flgA Class 2 flgB Class 2 flhB Class 2 fliA Class 3 fliD Class 3 flgK Class 3 fliC Class 3 meche Class 3 mocha Class 3 flgM
Activator of class 3 Master regulator
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Time [protein]
Induction of positive regulator
Promoters with decreasing affinity for regulator
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[protein]
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With the flagella regulon, current algorithms can distinguish Level 2 and Level 3 genes based on subtleties in expression patterns not readily distinguished by visual inspection. Using our methods for expression profiling (sensitive, good time resolution) we have been able to demonstrate more subtle regulation than previously described. Different mechanisms can give rise to different patterns- in this case temporal patterns arise by transcription hierarchies (I.e. Level 1 ‡ Level 2 ‡ Level 3) and by differences in binding site affinities within a level. “You can not infer mechanism from pattern.”
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Methods such as the one described here or DNA microarrays can be used to measure expression of all the genes in a cell simultaneously. Reverse Engineering challenge – can we use expression data to infer genetic networks?
E A D B C F U Z W Y X V M N O
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