Handout 4—Teacher’s Version 1. A fusion between PR and lacZ is used to monitor the role of CI repressor, OR1, OR2, and OR3 in expression of PR as a function of the repressor concentration in the cell. A separate fusion (in a different bacterial strain) between PRM and lacZ is used to monitor the role of repressor, OR1, OR2, and OR3 on expression of PR. The results of a typical experiment in which all genes and proteins are wild-type is shown below: a. Why does the activity of the PRM fusion goes up at the same time that the activity of the PR fusion goes down? Because of cooperativity between repressor dimers bound at OR1 and OR2, repressor will bind to those two operator segments nearly simultaneously. Therefore repression of P R and activation of PRM will occur to the same extent as repressor concentration is increased. P R turn-on will be accompanied by the shut-off of PRM. b. Why does the activity of the PRM fusion go down at very high CI concentrations? At high repressor concentrations, repressor binds to OR3. This shuts off PRM. c. Draw the curves you would expect to see if the cI gene contained a pc mutation? Superimpose your curves on the graph that was shown originally for part a of this question. The curve for PR should exactly parallel the curve shown in the original figure because repressor binding to DNA is unaffected by the pc mutation. However, the curve for P RM should start very low and stay flat because the promoter can’t be turned on by repressor. d. Draw the curves you would expect to see if the PRM promoter contained the “up” mutation prmup-1 and OR2 was made inactive (unable to bind repressor) by mutation. Superimpose your curves on the graph that was shown originally for part a of this question. The curve for PR should be displaced to the right somewhat because with OR2 mutated, the amount of repressor necessary to occupy OR1 will be increased since there will be no cooperativity between the two operator segments. For PRM, the up mutation means that activity will start out pretty high. There should be no activation because of the OR2 mutation, but at very high repressor concentrations there will be repression of P RM due to repressor binding to OR3. e. Cells are simultaneously infected by two mutant phages. One phage is cIpc, but otherwise wild-type. The other phage is OR2–, but otherwise wild-type. Should the two phages complement; that is, should they be able to form stable lysogens? Why or why not? No stable lysogens can be formed because the otherwise wild-type phage that has the pc mutation makes repressor that can’t activate PRM. Similarly, the OR2 mutant prophage might be expected to supply good repressor to allow repression of both prophages, but its PRM can’t be activated because repressor can’t bind to OR2. So neither phage is able to make repressor capable of activating PRM and maintaining the prophage state. 2. On mixed infection of non-lysogenic E. coli cells with pairs of lambda mutants (two different phages), some pairs of mutants can form lysogens and other pairs cannot. Which of the following pairs should form lysogens? Briefly explain your reasoning. (Assume phage genes not indicated are wild-type). a. λ cIII+ cI- and λ cIII- cI+ Here, each phage can supply the trans-acting protein that the other lacks. This is exactly how complementation occurs. In other words, lambda needs good CI protein and good CIII protein to lysogenize and both are present in this mixed infection. CIII protein will allow turn-on of PRE on both DNA templates; this will allow the good cI gene to be turned on, providing CI protein necessary for lysogenization. b. λ PRM+ cI- and λ PRM- cI+ Here, lysogenization cannot occur because in the maintenance phase of repressor synthesis, on phage can produce cI mRNA which encodes mutant CI protein, but the other phage, which has a good cI gene, cannot produce cI mRNA. This is an example of the inability of a cis-acting mutation (in PRM) to be complemented in trans. c. λ cIPC PRE+ and λ PRE- cI+ This is a harder example. Here, the establishment synthesis of CI is confined to the phage that produces cIPC repressor. So when it is time to turn on PRM, the mutant repressor can repress lytic functions, but cannot turn on PRM, so there is no repressor in the maintenance phase. Thus, no lysogen can be maintained. d. λ cI- OR2+ and λ cI+ OR2This is essentially identical to (b). OR2 is the activator (repressor) binding site necessary to turn on PRM. Therefore, mutations in OR2 are equivalent to mutations in PRM. They are cis acting, so they cannot be complemented in trans. Although repressor binding to OR1 could shut off PR, no repressor can bind to OR2, so PRM cannot be turned on. e. λ cI+ cII- and λ cI- cII+ Here, lysogenization can occur. Both cI and cII produce trans-acting proteins. During infection, complementation occurs. One phage supplies CII which can turn on repressor synthesis from PRE of the other phage, which then produces repressor necessary to establish lysogeny. f. λ cI+ PRE- and λ cI- PRE+ Here, lysogenization cannot occur. During the establishment phase of repressor synthesis, no repressor can be produced for reasons similar to (b). One phage can produce repressor mRNA, but no active repressor; the other phage can produce good repressor, but only if the gene is transcribed; if the gene is transcribed, but it cannot be because the establishment promoter is defective. No repressor during the establishment phase means that no lysogens can be produced. g. λ PRM+ PRE- and λ PRM- PRE+ Here, lysogenization can occur. During the establishment phase, the second phage can produce repressor; during the maintenance phase, the first phage can produce repressor. Sp a double lysogen can be established, and then maintained because the first prophage will make enough repressor to act in trans and shut off both infecting phages.