Chapter 17-Gene Control in Bacteria1. From DNA to RNA to Activationa. DNA---mRNA---protein----activated protein-gene expression can be controlled at any point here2. 3 Mechanisms for cell to avoid producing unneeded proteins1. avoid making mRNAs for certain enzymes-known as Transcriptional Control-occurs when regulatory proteins affect RNA polymerase's ability to bind to promoter.2. Cell might have way to prevent mRNA from being translated if it has been transcribed already-known as Translational Control-regulatory molecules shorted life-span of mRNA-so that it gets degraded by ribonucleases faster3. Some proteins need to be phosphorylated to become active-so this can be prevented in what is called Post-Translational ControlNote: All of these occur in bacteria3. Transcriptional Controla. saves most energy for the cellb. stops process at earliest possible pointc. slow reaction4. Translational Controla. allows a cell to make rapid changes in the proteins5. Post Translational Controla. provides most rapid response of all 3 mechanismsb. spends a lot of energy6. Gene expressiona. genes are not just on or offb. they can vary in their level of expression7. Metabolizing Lactosea. Key model of gene regulationb. E. Coli can use many sugars for ATP production-via cellular respiration and fermentationc. To use lactose E coli must transport the sugar into the cell-once lactose is inside, enzyme Beta-galactosidase catalyzes a reaction-that breaks the sugar down into glucose and galactoseDraw out experiment from p. 309d. researchers found that lactose regulates gene for Beta-galactosidase-lactose acts as inducer: a substrate in a reaction that stimulates expression of specific genes8. Beta-Galactosidasea. produced only when lactose is present and glucose isnt9. Identifying Genes Under Regulatory Controla. To find mutants associated w. particular trait there are 2 steps1. Generate large number of individuals w mutations in random locations-UV light, X-rays, mutagens-mutagens: chemicals that damage DNA and increase mutation rate2. Screen mutants to find individuals w. defects you are looking for10. Replica Plating to find Mutant Genes1. when mutants w. defects in lactose metabolism are desired-mutagenized bacteria are spread on master plate-filled w. gelatinous agar containing glucose.a. Bacteria allowed to grow into colony2. Block w sterilized velvet pressed to master platea. cells transferred to velvet3. Velvet pressed onto plate containing a mediuma. medium: a liquid or solid that supports growth-must differ from master plate by a single component4. After cells grow compare colonies that thrive on replica plate's medium with those on the master plate11. Indicator Platesa. Where mutants w. metabolic deficiencies are observed directlyb. Compound used by Monod is acted on by Beta-galactosidase-compound acts as indicator molecule for functioning Beta-galactosidase-bec. one of the molecules produced by this reaction is yellowc. colonies w. defect in Beta-galactosidase enzyme or its production turn white12. Different Classes of Lactose Metabolism Mutantsa. The initial mutant screen yielded three types of mutantsb. In one class the cells cannot cleave indicator molecule even if lactose is present as inducer-interpretation: No Beta-galactosidase: gene for it is defenctivec. Second class the cells cannot accumulate lactose-interpretation: no membrane protein to import lactose-gene for galactoside permease defectived. Third Class the cells cleave indicator molecule w/o lactose as inducer-interpretation: gene for regulatory protein that shuts down lacZ is defective13. Constitutive Mutantsa. Abnormal cells that produce a product at all timesb. Mutated gene that produced constitutive Beta galactosidase was named lacI-cells w mutant form of this, gene expression occurs w or w/o lactosec. In these mutants gene remains on when it should be off14. Putting Observations Togethera. hypothesized that normal product of lacI gene-prevents transcription of lacZ and lacY when lactose is absentFigure 17.415. Several Genes Involved in Lactose Metabolisma. 3 genes involved in lactose metabolism-lacZ lacY and lacIb. lacZ and lacY code for proteins req. for metabolism and import of lactose-lacI responsible for regulatory functionc. These 3 genes are close together-suggests that lacZ and lacY might be controlled by lacI16. Regulation of Transcription Figure 17.6a. 2 possible ways- Negative Control and Positive Controlb. Negative control -occurs when regulatory protein binds to DNA and shuts down transcriptionc. Positive Control-occurs when a regulatory protein binds to DNA and triggers transcriptiond. lacZ and lacY under positive and negative control17. Repressors Figure 17.7a. lacI gene produces an inhibitor-exerts neg. control over lacZ and lacY genes-transcription inhibitor called a repressor-binds directly to DNA or near promoter for the genesb. Lactose interacts w repressor-causes repressor to release from binding sitec. Repressor is "parking brake -Lactose releases "parking brake"18. Constitutive Mutantsa. E coli cells w functioning copies of genes for B-galactosidase-and galactoside permease-but lacks functional gene for repressorb. Result: They make B-galactosidase all the timec. When the cells are given a functional copy of repressor gene-B-galactosidase production declines and stopsd. So, repressor codes for protein that shuts down transcription19. So What?a. lacI gene codes for a repressor protein -that exerts negative control on lacZ and lacY.b. Lactose is inducer by removing repressor and ending neg. control.20. The lac Operona. Operon: set of coordinately regulated bacterial genes-that are transcribed together into one mRNA.b. Group of genes involved in lactose metabolism termed lac Operonc. lacA gene codes for enzyme transacetylase-catalyzes reactions that allow certain types of sugars to be exported from the cell when they are too abundant21. 3 Central Hypotheses to Jacob-Monod model of lac operon regulation1. lacZ, lacY, lacA are adjacent -and transcribed into one mRNA initiated from single promoter of lac operon2. repressor is a protein encoded by lacI-that binds to DNA and prevents transcription of lacZ lacY and lacANote: section of DNA in lac operon where repressor binds-is called the operator.3. Lactose binds to repressor-repressor changes shape-and causes repressor to drop from DNA strandThis is another form of allosteric regulation22. Catabolite Repressiona. Transcription of lac operon-is
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