Slide 1Not All DNA Codes for ProteinsWays to Measure Gene ExpressionCount the Messenger RNA MoleculesEnzymatic Assay or In-Situ HybridizationEnzymatic Assay or In-Situ HybridizationSlide 7Simple Case of Turning a Gene “On”Slide 9Measuring mRNA & protein numbersSlide 11Transcriptional bursting in eukaryotesThe Lambda Switch: The Other Hydrogen Atom of Gene RegulationBacteriophage and Their GenomesSlide 15The Life Cycle of Bacteriophage LambdaA Genetic SwitchThe Lambda GenomeA More Detailed Example of the Parrts ListThe Lambda Switch: Lysogenic StateThe Lambda Switch: Lytic StateDNA Geography of the SwitchBinding of Transcription FactorsSynthetic Genetic SwitchStable SolutionsPhase Portrait for the SwitchSynthetic Genetic SwitchSlide 28Slide 29Slide 30Coupling of Genes in NetworksThe Central Dogma of Molecular Biology: How Genes Lead to Proteins(Berman et al.)Crick and others mused over the ``two great polymer languages’’. Central dogma explains the chain of events relating them.The ribosome is the universal translating machine that speaks both languages.We have seen what genes are and how they serve as the informational memory of organisms. But we have NOT said how they are controlled.Now we have the background to tackle the question we started with: how do cells make decisions?Not All DNA Codes for Proteins(Berman et al.)The regulatory landscapeThe E. coli genome is a circle with roughly 4.7 million base pairs.How many genes? An estimate.The genes related to sugar usage have been one of the most important stories in the history of modern biology and biochemistry (and take us right back to the great debate on vitalism played out with Pasteur in the 1800s).“Promoter” region on DNA is subject to intervention by various molecular bouncers that govern the gene.Ways to Measure Gene ExpressionBasic point: looking for “reporters” of the level of expression of gene of interest.Can ask the system to report on the level of gene expression at various steps in the processes linking DNA to active protein.Promoter occupancy, level of mRNA, level of active protein.This image shows a Drosophila embryo colored to show the expression patterns of early gene regulators. Each color represents the level of expression of one of three gene regulators, Knirps (green), Kruppel (blue), and Giant (red). Color intensity reflects a higher level of expression. The darker areas of the embryo are cells where none of these gene regulators are expressed, and the yellowish areas indicate that both Knirps and Giant are being expressed.http://www.lbl.gov/Science-Articles/Archive/sabl/2008/Feb/genome-mystery.htmlCount the Messenger RNA MoleculesEnzymatic Assay or In-Situ HybridizationEnzymatic assays – promoter leads to the production of a protein that then does some enzymatic action on the substrate which yields a product that can be visualized.In-situ hybridization – described the other day – probe is complementary to the RNA of interest and is labelled for detection.Enzymatic Assay or In-Situ HybridizationEnzymatic assays – promoter leads to the production of a protein that then does some enzymatic action on the substrate which yields a product that can be visualized.In-situ hybridization -Ido GoldingInformation Processing in Living Cells:Beyond First ApproximationsCaltech 11/2008Department of Physicsts(t)Approximations used to describe the process…tr(t) Stimulus (sugar)Response (RNA production)Simple Case of Turning a Gene “On”Plac/ara RFP 96x MS2-bsGene of interest:Engineering bacteria to report on gene activityGolding et al., Cell (2005) IPTG, arabinoseRNA targettranscription(RNA-tagging protein;in excess in the cell)MS2-GFPbindingRFP proteintranslationMeasuring mRNA & protein numbersmRNA number of bound MS2-GFPs photon flux from localized green fluorescenceProtein number of RFPs photon flux from whole-cell red fluorescenceHistogram of RNA copy number:1st peak = inter-peak interval 50-100 X GFP = 1 transcriptControls:QPCRProtein levelsLux: Lutz & Bujard 1997Controls:FISH(Thanks to: A. Raj, A. van Oudenaarden)0 2 4 600.10.20.30.4Number of RNAProbability n, 95 events0 20 40 60 8000.050.1Time (min)Probability of, 117 eventson, 95 events051015mRNA051015mRNA051015mRNA0 50 100 150 200 250051015Time (min)mRNADistribution of burst sizeDistribution of on & of times# mRNA vs timeRNA kinetics in individual cellsTranscriptional bursting in eukaryotesChubb JR, Trcek T, Shenoy SM, Singer RH. Curr. Biol. (2006)See also: Golding & Cox, Curr. Biol. (2006)Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S, PLoS Biol. (2006) “Stochastic mRNA Synthesis in Mammalian Cells”.The Lambda Switch: The Other Hydrogen Atom of Gene Regulation Roger HendrixBacteriophage and Their Genomeshttp://www.biochem.wisc.edu/inman/empics/0020b.jpgPhysical Consequences of the Tight Squeeze in the Life Cycle of a BacteriophageRate of packing: 100bp/sec“Some assembly required”Self-assemblyRate of ejection: ≈ 100 - 1000bp/secConstruct a physical model of these processes.Forceful ejectionThe Life Cycle of Bacteriophage LambdaA Genetic SwitchThe Lambda GenomeA More Detailed Example of the Parrts ListThe Lambda Switch: Lysogenic StateThe Lambda Switch: Lytic StateDNA Geography of the SwitchBinding of Transcription FactorsSynthetic Genetic Switch Collins et al. - see course websiteStable SolutionsPhase Portrait for the SwitchSynthetic Genetic Switch Collins et al. - see course websitea, Snapshots of phase-contrast image showing cell F and its progeny and b, related bioluminescence image at different times t (given in days, a 24 h period of time) from the beginning of the measurement. Pixels in the bioluminescence images were binned 3 times 3 (pseudo-colour, where red is high signal intensity and blue is low signal intensity). Scale bar, 5 microm. c, The size of the cell F and all its progeny as a function of time measured from the phase-contrast images (non-binned pixels). The arrows point to the time where the snapshots in (a) and (b) were taken. d, The total number of pixels occupied by F and its all progeny versus time (black line) plotted in a logarithmic scale. The red line is the corresponding exponential growth fit: total size (t) = initial size times 2t/tau with tau = 23.04 plusminus 0.17 h. e, Density of bioluminescence for the same cell and all its progeny versus time. f, The average density of bioluminescence versus time (black line) and its fit (red line) with: left
View Full Document
Unlocking...