Questions–TwoComponentSystems - Questions...

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Unformatted text preview: Questions to Two Component Regulatory Systems and Environmental Sensing 1. The addition of glycine to the growth medium of B. lictus results in increased transcription of the glyC gene. A mutant strain of B. lictus that has a loss-of-function mutation in the glyR gene expresses higher levels of glyC than the wild-type strain in the absence of glycine. There is no difference in glyC expression in the presence of glycine. This data is represented in the following graph: A) The protein encoded by glyR is involved in transcriptional [activation/repression] of glyC. B) What experiment would you perform with purified GlyR protein to determine whether it could directly control transcription of the glyC gene? Please indicate the key components that would go into this experiment. (Note: you do not need to indicate what potential results would be, but the experimental set up should include a control.) 2. If a two-component regulatory system is activated by a small molecule, that small molecule most likely binds to the [kinase/response regulator] component. 3. In response to an activating environmental signal, what component of the two-component regulatory system becomes phosphorylated? a) kinase b) response regulator c) both the kinase and response regulator 4. Transcription of the ureA gene of V. crestus is induced by the presence of urea. In mutant strains of V. crestus that lack either the regA or the regB gene, the ureA gene is not induced by the presence of urea. You hypothesize that the regA and regB genes encode a two-component regulatory system that activates transcription of ureA. Using purified RegA and RegB proteins, describe an experiment using an “in vitro transcription assay” to test your hypothesis. Be sure to include appropriate controls. Describe expected results if your hypothesis is correct. 5. Name the two proteins that comprise a “Two-component regulatory system”. 6. You are studying how the bacterium, Rhizobium meliloti, senses acidic pH stress. A gene, phrA, is activated by acidic pH stress. In a mutant strain that lacks the protein PhrR, phrA is not transcribed either in the presence and absence of acidic pH stress. You hypothesize that PhrR is a transcriptional activator that binds to the promoter region of phrA and activates transcription. Propose one experiment to test this model. Include appropriate controls. List two potential results and what you would conclude from each. (12 points) 7. Describe and/or draw the general features of a two-component regulatory system. 8. You are studying regulation of the narG gene by O2 in the bacterium, V. curvius. Transcription of this gene is higher in the absence of oxygen, as determined by measuring ß- galactosidase specific activity of V. curvius cells containing a narG-lacZ transcriptional fusion. You have isolated a mutant, called the NarR- mutant, that has altered expression of narG-lacZ in response to oxygen. The NarR- mutant lacks the NarR protein. You grow wild-type and NarRmutant cells, both of which contain narG-lacZ, in the presence and absence of oxygen and measure ß-galactosidase specific activity. The following graph shows the results obtained. A) From these results, list the conclusions that you can draw about the role of NarR in regulating transcription of narG in response to O2. 100 80 60 Absence of Oxygen 40 Presence of Oxygen 20 0 Wild-Type NarRMutant B) You hypothesize that NarR is a transcription factor that binds to DNA around the promoter of narG and affects transcription. You purify the NarR protein. Describe the outline of ONE experiment to test this hypothesis. Indicate TWO controls that should accompany this experiment. C) From the amino acid sequence of NarR, you find that find that the NarR protein is similar to Response Regulator proteins. i) Draw the general organization (i.e. domain structure) you expect for the NarR protein. ii). Indicate the expected location and name of any amino acids that are known to be critical for the function of Response Regulator proteins. iii) Draw a model for the mechanism for activation of activation of the NarR protein based on your knowledge of how Response Regulators are activated. 9. You are studying how PO4- limitation is sensed by G. roundus. PO4- limitation results in increased transcription of the gene alkP. You identify several regulators that affect the expression of alkP and propose the following model for how these regulators affect transcription of alkP. At low [PO4-], the Atr protein is stimulated to methylate the Map protein. When the Map protein is methylated, it stimulates the phosphatase activity of the KinA protein. KinA dephosphorylates RegB. Unphosphorylated RegB is unable to bind to DNA, and transcription of alkP proceeds. At high [PO4-], the Brp protein is stimulated to remove methyl-groups from the Map protein. Unmethylated Map protein is inactive. KinA acts as a kinase to phophorylate RegB. RegB binds to the promoter region of alkP to inhibit transcription. From these models, indicate in the table below what phenotype you would predict for the following mutants for alkP transcription at high and low [PO4-]. Indicate whether you would predict transcription of alkP to be “high” or “low”. Also, provide a brief explanation of why the mutant has the phenotype that you predict. PO4- Concentration Phenotype of mutants Explanation High Low Wild-type Low High Lacks the Atr protein Lacks the Brp protein Lacks the Map protein Lacks the KinA protein Lacks the RegB protein ...
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This note was uploaded on 03/06/2012 for the course MIMG 100 taught by Professor Lazazzera during the Summer '10 term at UCLA.

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