BE/APh161 – Physical Biology of the Cell!Rob Phillips Applied Physics and Bioengineering California Institute of TechnologyHow Cells Decide as Seen Through Three Hall of Fame ``Model’’ Organisms!(Berman et al.) E. coli yeast Fruit fly ! Various organisms are accorded hall of fame status as ``model’’ organisms either because they are specialists at some particular process of interest or they are experimentally convenient (grow fast, easily accessible). ! Each of these organisms offers something extremely important on the question of how cells decide.The 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?The Big Message!The Puzzle: All the cells in a given organism (almost) carry the same genetic information. And yet, depending upon where they are within the organism, they turn out quite differently. The Insight: The genome (i.e. genetic material) is under exquisite control. Genes are turned on and off in response to environmental cues. This lecture: how we found out, some beautiful examples, where we stand now.Measuring the Diet of a Bacterium!! Growth curves have served a central role in dissecting the physiology of cells of all types. ! In particular, we know much about how cells decide based upon watching them grow and seeing what they like to eat.Deciding What to Eat: Giant Discoveries Often Arise From Seemingly Arcane Topics!(Berman et al.) ! Fascinating twist of history of science: human curiosity leads to investigation of seemingly arcane topics (spectral lines of atoms, specific heats of solids, peculiarities in the orbits of Uranus or Mercury, etc.) from which emerge hugely important insights. ! An example: nutrition of single cells like yeast and bacteria. ! Yeast cells express preferences about which sugar to use. ! Interestingly, the proteins used to digest the less preferable sugars are only synthesized when those sugars are present and the more preferable sugars are absent. (Spiegelman et al., PNAS, 1944)A Model System for Mathematically Dialing in Transcription!(Berman et al.) ! The way all of this works was first figured out in the context of a very specific question in bacteria. How do cells implement the decision that they prefer some sugar sources (i.e. glucose) over others (i.e. lactose)? ! What emerged was a picture in which genomic DNA is controlled by an army of molecular bouncers (transcription factors) that activate or repress expression of their genes of interest. Bacterial growth curvesRepressors: The Cartoon!! Repressor molecules inhibit action of RNA polymerase. ! Repressors can be under the control of other molecules (i.e. inducers) that dictate when repressor is bound and not.Activators: The Cartoon!! Activator molecules enhance the action of RNA polymerase. ! Activators can be under the control of other molecules (i.e. inducers) that dictate when activator is bound and not. ! Activators “RECRUIT” the polymerase. Adhesive interaction between RNAP and activatorNot All DNA Codes for Proteins!(Berman et al.) The regulatory landscape ! The 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.Lac Operon: The Single Molecule Census!(Beautiful work of David Goodsell)The Lambda Switch: The Other Hydrogen Atom of Gene Regulation !Roger HendrixBacteriophage and Their Genomes!http://www.biochem.wisc.edu/inman/empics/0020b.jpgRate of packing: 100bp/sec “Some assembly required” Self-assembly Rate of ejection: ≈ 100 - 1000bp/sec Forceful ejection Bacteriophage Life cycle 1!The Life Cycle of Bacteriophage Lambda !A Genetic Switch !The Lambda Genome !The Lambda Switch: Lysogenic State !The Lambda Switch: Lytic State !DNA Geography of the Switch !Binding of Transcription Factors !Measuring fold change: The Cell as a test tube!! Install the architecture of interest in the cell and then “read out” the state of the DNA and its battery of attendant proteins using gene expression.Enzymatic Assay or In-Situ Hybridization!! Enzymatic 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 Hybridization!! Enzymatic 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 -Ways to Measure Gene Expression!! Basic 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.htmlA Standard Candle for Gene Expression!Hubble Space Telescope Cepheids variables ! A prerequisite for doing the theory-experiment comparison in the way advocated here is that one has to really know the meaning of the
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