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Lecture 15

Lecture 15 - Prokaryotic Gene Regulation

5 Pages
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Department
Biology
Course Code
Biology 2581B
Professor
Jim Karagiannis

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LECTURE 14 PROKARYOTIC GENE REGULATIONKey Concepts1Catabolite Repression2Molecular Basis of Genetic Control3Trp Operon4AttenuationOperon Model RepressionIn the absence of lactose the repressor binds to the DNA of the operator and this binds to the DNA of the operator and this binding prevents transcription The repressor thus serves as a negative regulatory element Operon Model InductionBased on their genetic analysis Jacob and Monad developed a simply yet highly detailed model of inductionElement of ModelKey ResultsA repressor protein coded for by the lacI gene Loss of function mutations in lacI lead to constitutive expressionrepresses transcription in the absence of lactoselacI gene product acts in transcThe repressor protein inhibits transcription by binding lacO mutations lead to constitutive expressionto a DNA sequence adjacent to the promoter clacO acts in cisoperator and blocking RNA polymerase bindingInduction results from binding of inducer allolactose Under specialized circumstances PaJaMo expt induction is to repressor preventing the repressor from binding possible in the absence of inducerthe operatorSlacI mutants that are unable to bind allolactose are noninducibleWhen lactose is present allolactose an inducer derived from the sugar binds to the repressor This binding changes the shape of the repressor making it unable to bind to the operatorWith the release of the repressor from the operator RNA polymerase gains access to the lac operon promoter and initiates transcription of the three lactoseutilization genes into a single polycistronic mRNADNA FootprintingDNA footprinting experiments establish the region of DNA to which a protein bindsGoal of DNA fingerprinting is to establish region of DNA to which a protein bindsTo do this experiment you need a DNA fragment and what you do is you add a radioactive phosphate to the 5 end which allows you to track where each of these fragments are going in the experimentTake this labeled fragment and split it into different samplescontrol does not consist of punitive binding protein whereas your other sample doesEach sample is digested with DNase 1 not a restriction enzyme so it does not recognize specific sequences which cuts randomly along the DNA fragmentAfter digestion you end up with a whole series of fragments of different sizes which are collected and run on an agarose gel where they are separated according to size An xray film is laid on top of the gel and wherever there is radioactivity present a black band becomes visible on the filmFor the control there is a separation from all the different bands that are generatedBut in the presence of a protein when a protein binds it covers up and protects the sequence so where it is bound the DNase 1 is not going to be able to cutThus a whole series of fragments are missing because they are protected by the binding which is referred to as a footprint in the gelindicates directly that the protein is able to bind the DNA fragmentSee image on the next page top right
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