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BIOL 308
Dragana Miskovic

STUDY QUESTIONS: LEC 18-19 1. You have discovered base changes in the promoter region of the operon in a bacterial chromosome. Would you expect these changes to act in trans on another copy of the operon? Explain your reasoning. The changes in the promoter region are cis-acting because the promoter is a binding site and does not have a gene product that can move to affect another copy of the operon. 2. What are cis- elements? What are trans- factors? Give an example from the Trp operon (or form the Ara-operon). Cis-elements are regions of DNA/RNA that generally affect processes happening on the same strand of DNA. For example, in the Ara-operon, the araO is2an operator that when bound to AraC dimer, blocks RNAP from binding and thus inhibits transcription. Thus, it acts in cis by affecting the events taking place in the region of the DNA. In the Trp operon, the operator is also like a cis-element since the repressor dimer (bound with tryptophan) can bind it and transcription of the Trp genes is inhibited. Trans-factors are proteins that can be produced somewhere else in the cell and diffuse through the cell, binding to regions of the DNA and influencing transcription. In the ara-operon, AraC is a trans-factor since it is produced by one set of genes and can diffuse and act on the ara genes. Also, CAP is a trans-factor since it is produced somewhere else in the cell and can bind promoters in the presence of cAMP and enhance transcription of catabolic genes. 3. Draw the diagram of the lac operon that illustrates negative control (be careful here and think about the complete picture!!!!!). lacR is produced and binds the lac operator when lactose is absent, preventing transcription. Lactose acts as a co-repressor or inducer by binding lacR, keeping it from binding the operator and thus allowing transcription. This lactose-lacR combination also works to inhibit lacR synthesis, through ‘autoregulation’. 4. You have isolated a protein that binds to DNA in the region upstream of the promoter sequence of the gene of interest. If this is a positive regulator (activator) which would be true: A) Loss of function mutation in the gene encoding this DNA binding protein would cause constitutive expression B) Loss of function mutation in the gene encoding this DNA binding protein would result in lower or no expression. Explain your reasoning. Lower or no expression (B) would be expected because normally, an activator is produced that binds the enhancer, increasing the ability of RNAP to bind and for transcription to be initiated. So if the activator is not available, the gene of interest would be expressed less. 5. Discuss why are lac O mutants cis-acting. These mutants are cis-acting because the operator within that region of DNA is mutated, preventing lacR from inhibiting transcription. If a merodiploid situation is introduced, this mutated operon will not affect the expression of the plasmid lac operon, which will be repressed by lacR as normally. 6. Discuss why are lac I mutants trans-acting. These mutants are trans-acting because the repressor is a protein that is produced and can diffuse through the cell. Thus, if non-inducible lacR is produced, it will bind lac operons throughout the cell, even if they are on a plasmid. 7. Discuss positive and negative regulation of L-ara operon. Positive Regulation: - In the presence of arabinose, it binds to AraC (activator now), this causes a change in conformation of the protein so AraC binds to Ara I and 1ra I and n2t to ara O2, P BAD promoter is open and transcription starts. Also activated through CAO-cAMP pathway. Negative Regulation: - In absence of arabinose, AraC maintains its original conformation. It binds to ara O 2nd ara I ,1this causes the DNA to loop and hides the P BAD promoter from RNAP. Transcription is inhibited. In addition, when AraC is too plentiful, AraC binds with AraO to have promoter blockage. 8. Regarding the regulation of Trp operon, what do we call the amino acid tryptophan? Why? The amino acid tryptophan is a co-repressor. This is because it binds the aporepressor TrpR, which is normally not able to repress the trp genes, and allows it to then inhibit transcription of the trp genes. Because trp itself is not repressing transcription directly, and because the aporepressor requires it for repression, the amino acid tryptophan acts as a ‘co-repressor’. 9. What is meant by polycistronic mRNA? Give an example. Polycistronic mRNA means that there are several genes under the influence of one promoter. An example is in the lac operon, where beta-galactosidase, permease, and the lacA product are all under control of one promoter. Initiation of transcription at the promoter will allow all genes to be present on the mRNA product. Translation of this cluster of genes can then take place, allowing the important components of the processes to be present. 10. What is catabolite repression? What is the role of Catabolite Activator Protein? Explain its action (remember the # of operons it activates!). Catabolite repression is the effect observed when glucose is present in a cell  because glucose is favoured for metabolism, transcription of other catabolic genes will be repressed as they do not need to be on. CAP is activated by cAMP, which is produced during low levels of glucose – it works by activating over 100 catabolic genes including the arabinose and lactose genes, thus allowing alternative forms of energy to be utilized. 11. Define: repressor, co-repressor, aporepressor and inducer. A repressor is a molecule that can inhibit transcription. A co-repressor is a molecule that can activate an apo-repressor, forming a repressor that can inhibit transcription. The co-repressor cannot have inhibitory effects on its own, and the apo- repressor is unable to be inhibitory without the co-repressor bound. An inducer is a molecule that induces gene expression by binding to a repressor, preventing it from binding DNA and having inhibitory effects. 12. Define effector and inducer. Give examples. Effector: A molecule that binds to trans factors either activating them or inhibiting their function and thus they can up or down regulate gene expression (Rep
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