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UIUC MCB 250 - 26-lac operon 2

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Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Self Doubt WorkshopWednesday 10/29/2014Lincoln Hall Room 10647pmThinking about pursuing healthcare but not exactly sure? Have your questions answered as we address and discuss the many doubts and fears that everyone faces in their college career.MCB 320 - spring semesterMechanisms of Human DiseaseAdvanced MCB credit - 3 semester hoursOverview:Instructors:Eric Bolton, Ph.D. [email protected] Llano, M.D., Ph.D. [email protected]:Undergraduate Juniors and Seniors with biomedical interestsPrerequisite:MCB 252 or instructor consent Coleman and Tsongalis, 2009, Molecular Pathology: The Molecular Basis of Human DiseaseLearn how molecular and cellular defects manifest as pathologies that affect the function of human tissues and organs. This course focuses on the pathophysiology of common human diseases and the environmental, genetic, and epigenetic causes of specific disease types. Disease topics include cardiovascular diseases, stroke, cancer, asthma, COPD, diabetes, metabolic syndrome, neurologic disorders, and reproductive disorders.Transcriptional regulation in bacteria (continued)Lecture outline1. Review the lac operon✦Functional analysis using genetic mutants✦Concepts in gene interaction: cis vs. trans regulation ✦Interaction between multiple operators.2. Coordinated expression of multiple genes✦A single transcription factor can regulate many genes in parallel.✦Different sigma factors can direct RNA polymerase to different sets of genes.+1lacOlacZ lacY lacAlac PromThe lac operonCAP siteTranscriptional regulation of the lac operon is accomplished by three proteins (i.e. RNA poly-merase and two tran-scription factors) with distinct DNA binding sites.Jacob and Monod figured out the mechanism of lac operon regulation by studying genetic mutations.The nomenclature of functional genetics:•The normally functioning or wild-type allele of a gene is often designated with a '+': e.g. when expressed, the allele lacZ+ produces a functional b-galactosidase protein.•Loss-of-function mutations are often designated with a '-': e.g. lacZ- refers to a copy of this gene that is incapable of producing a fully functional -galactosidase protein. Note: some mutations produce a partial loss of function, whereas others (called ‘null’ alleles) cause a complete loss of function. In MCB250 we will assume loss-of-function alleles are null unless specifically stated otherwise.A second tool that Jacob & Monod used to study the lac operon was the F plasmid: •Plasmids are self-replicating, typically circular dsDNA molecules that can coexist with the bacterial chromosome. •Plasmid DNA will sometimes exchange gene(s) with the bacterial chromosome.•Plasmids can be transferred from one cell to another.Watson, Fig. A-6F+Jacob and Monod isolated E. coli in which part or all of the lac operon had recombined onto the F-plasmid, then got this recombinant plasmid to transfer into other bacteria. The recipient bacterium was effectively diploid for these genes, allowing the researchers to study how different parts of the operon interact with one another.Watson, Fig. A-6Watson, Box 18-2, Fig. 1A lacI gene on one piece of DNA can repress the lac operon on the same DNA or a different chromosome as long as they occupy the same cell.Cis vs. Trans regulationIn genetics, two elements are said to function "in cis" if they must be on the same DNA molecule to interact. Example: a promoter and its transcription unit only function in the cis arrangement. A promoter cannot be used to transcribe a gene on another chromosome.In contrast, two elements are said to function "in trans" if they can interact when located on the same or different DNA molecules.Example: once translated by a ribosome, a protein can potentially interact with any DNA molecule in the cell.+1lacOTranscriptionlacIlacZ lacY lacAlac PromlacI PromDNA sequences show cis regulation, i.e. they only control the expression of genes located on the same DNA molecule.TranscriptionTranslationCAPCAP PromTranslationCAP siteCis regulatory elementsTrans regulationThe functional LacI repressor protein is actually a tetramer capable of binding two different operator sequences simultaneously.Subsequent to Jacob and Monod, it was discovered that the lac operon has two additional operator sequences, each located less than 500 base pairs away from the primary operator (O1).Watson, Fig. 18-12(LacI)lacZ(O1)(O3)(O2)-foldLacI bindingaffinity: O1>O2>O3Strength of repression:The significance of interactions between operators can be seen by comparing the level of repression when one or more operators have been deleted.REPRESSION(LacI bound to DNA)NO REPRESSION (LacI not bound to DNA)Compare transcription factorsThe LacI repressor and CAP activator proteins have several common features:•Helix-turn-helix protein structures.•Bind to specific DNA target sequences (although their specific target sequences differ).•Allosteric regulation of DNA binding affinity by a small intracellular ligand. Presence or absence of each ligand reflects the state of sugar metabolism.•The transcription factor physically interacts with RNA polymerase to influence the rate of gene transcription.And an important difference …The LacI repressor protein only influences the expression of a single transcription unit, the lac operon.In contrast, there are hundreds of E. coli genes that contain CAP binding sites and whose transcription is regulated by CAP.***********GlucoseLow intracellular [cAMP]CAP inactive (= bind DNA)(preferred nutrient)Lactose operonMaltose operonGalactose operonArabinose operonetc.reduced transcription ofBecause transcription factors like CAP function ‘in trans’, the expression of one protein can simultaneously regulate tens or hundreds of genes as long as they possess an appropriate binding site adjacent to their core promoter.Alternative sigma (w) factorsIn bacteria, a


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