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University of Waterloo
BIOL 308
Dragana Miskovic

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. No, promoter is a cis element, it only affects the copy its encoded on. This is because, it doesn’t involve a protein that can diffuse to another site but the gene itself which is fixed to the DNA. Also, since the genes on the promoter with the defect will not be transcriped, they will not affect other operons in trans. 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 regulate the expression of genes on the same strand (Usually short inverted repeats). They are binding sites for trans factors. E.g. Cis element of L-araOP is enhancer where the activator binds to increase RNAP binding to promoter. Other examples would be TrpO and enhancers. Trans factors are proteins that bind to specific parts of DNA regulating their expression. They can diffuse and act on sites on either of the strands. E.g. Trans factor of TrpOP is aporepressor and corepressor that bind to operator to inhibit transcription of TrpR and tryptophan and AraC of Ara. 3. Draw the diagram of the lac operon that illustrates negative control. Inducer gene located before structural ones is transcribed before and binds to the operator. If lactose is present, inducer binds to it so that it cannot bind to operator anymore. Inducer gene has its own promoter, termination sequence. 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. B. Since the protein is an activator, its absence will cause loss of activation and therefore, loss of positive regulation. This will lead do a decreased rate of expression. In the case of cAMP-CAP complex, loss of the complex will prevent the bending of DNA and therefore, the binding of the RNAP to the promoter site will be weak and the rate of transcription will be low. 5. Discuss why are lac O mutants cis-acting. LacO is having constitutive defect with the operon’s operator. It is only cis acting as trans factors would not be able to bind to this particular operon but would be able to do so for other operators of other operons. Operator is also an element, so it cannot travel. - 6. Discuss why are lac I mutants trans-acting. I is dominant over I and I is dominant over all. Such mutations can be either constitutive (product continuously formed) or non-inducing (product no longer formed). I permanently blocks the operator while I binds to the inducer to inhibit the process of transcription. LacI are trans acting as this produces repressors that are trans factors. Factors, unlike elements, are able to diffuse through to affect other operons. This defect causes inability to produce the factor and operators on its and other operons that depend on its binding for functionality won’t be able to work. 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 I1and Ara I 2nd not to ara O2, P BADpromoter 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 O2and ara I1, this causes the DNA to loop and hides the P BADpromoter 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? Corepressor. Because it binds to the aporepressor, activating it and causing a reduction in the transcription rate. It requires aporepressor to function. Also, autoregulator. Because in inhibits transcription of operon and transcription of TrpR gene as well. 9. What is meant by polycistronic mRNA? Give an example. Polycistronic mRNA means that from the same mRNA, many different proteins may be produced. It is transcribed from multiple genes. This is possible for prokaryotes as their structural genes within an replicon or operon have their own stop and start sites. An example is LacZ (beta galatosidese), LacY (permease) and LacA (trans acetylase) within lactose operon that have their own start and stop sites and produce different proteins. 10. What is catabolite repression? What is the role of Catabolite Activator Protein? Explain its action. Catabolite repression is repression caused when a substance with better energy producing ability appears. It represses usage of less energy efficient substrate (ex. glucose effect on lactose catabolism) CAP acts when it binds with cAMP to bind to enhancer elements where it increases RNAP activity. CAP also bends the DNA sequence to help RNAP binding and promote formation of open complex. RNA and trans factors need to be open in order to them to bind. Closed complex does not allow binding. 11. Define: repressor, co-repressor, aporepressor and inducer. Repressor: A trans factor. It’s a DNA binding protein that regulates the expression of one or more genes by decreasing their expression. It blocks transcription initiation when bound to the operator. Co-repressor: An effector molecule. It’s a protein that decreases gene expression by binding to a transcription factor which contains a DNA binding domain. Aporepressor: A trans factor. It’s a DNA binding protein that requires the binding of a corepressor (effector) to be able to bind to the operator region and decrease gene expression. Inducer: An effector molecule. It’s a protein that binds to the repressor inhibiting its function and thus, inducing gene expression. 12. Define effector and inducer. 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. They are (repressor/inducer and corepressor/aporepressor) Inducer: An effector molecule. It’s a protein that binds to the repressor inhibiting its function and thus, inducing gene expression. 13. What are activators? What are enhancers? Activators are regulatory proteins that bind to a site near promoter and enhance transcription. Enhancers are cis elements that increase transcription by influencing binding affinity of RNAP with promoter. They are binding sites for activators. 14. What is the role of auxiliary operators? Auxiliary operators increase the efficiency of primary operators by providing more sites for regulatory proteins to bind to. It also increases local concentration of repressors so that they may bind to the functional ones. For LacOP, it increases the repressiveness of Operator-Repressor binding effect by multiple folds. 15. Discuss the type of regulation of gene expression by two-component regulatory systems in bacteria? The two components are sensor-transmitter and response regulatory protein. A type of regulation done by this involves sensing the environment, causing the kinase transmitter to be autophophorylated and pass on its phosphate to response protein so that the protein can act as a trans factor to activate the effector domain and cont
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