Workshop 7
Q1. Regulators of the Grow1 gene
From a deletion analysis of the regulatory region of the Grow1 gene, you identify a 12bp palindromic sequence (G1) that is important for regulation of the Grow1 gene. You identify 2 new transcription factors that bind this G1 sequence – GA and GB – which have almost identical amino acid sequences in their DNA-binding domains , but little homology to each other elsewhere in the protein. GA is 400 amino acids long. GB is 250 amino acids long.
1.1 What does a palindromic sequence in the G1 binding site suggest?
1.2 What does the amino acid conservation between the DNA-binding domains of GA and GB suggest?
1.3 You clone 4 copies of either a wild type G1 site or a mutant G1 (G1m) site upstream of a TATA box and a luciferase reporter gene to make the plasmids G1-luciferase or G1m-luciferase. You then co-transfect either G1-luciferase or G1m-luciferase reporter genes along with increasing amounts of a plasmid to express either GA or GB. The data are shown below.
What can you conclude from these data about the function of GA and GB?
1.4 You then fuse the strong transcriptional activation domain from the VP16 viral transcriptional activator to GB, and co-transfect increasing amounts of GB-VP16 with either G1-luciferase or G1m-luciferase reporter genes. The data are shown below.
What can you conclude about the function of GB from these data along with the data above?
1.5 Finally, you transfect a standard amount of GA and increasing concentrations of GB with either G1-luciferase or G1m-luciferase reporter genes. The data are shown below.
There are several different explanations for these data – perhaps as many as 5. Describe 2 different models that are consistent with the data, and describe an experiment that would test one of the models that you have proposed. What would your experiment show if the model is correct? And what would it show if the model is incorrect?
Q2. Regulated activation domains
Signaling pathways that increase cAMP activate PKA, which then moves to the nucleus and phosphorylates and activates the CREB transcriptional activation domain. Activated CREB then increases transcription of c-fos and other genes.
2.1 If you add a protein synthesis inhibitor at the same time as increasing cAMP, will c-fos mRNA and protein levels still increase?
2.2. c-fos transcription is also activated by growth factors that activate a different signaling pathway that activates the MAP Kinase protein kinase. Activated MAP Kinase moves to the nucleus (like PKA), where it phosphorylates the SAP1 transcriptional activation domain and converts SAP1 from an inactive to an active transcriptional activator.
You are studying the SAP1 activation domain and make an expression vector that has DNA encoding the Gal4 DNA-binding domain (DBD) fused to the SAP1 activation domain. You transfect this Gal4-SAP1 expression vector into fibroblasts in culture with a reporter gene with Gal4 binding sites, and a co-transfection control. You do this for 2 plates – one to which you add growth factors that activate MAP Kinase, and one to which you do not add any growth factors. Your labmate says that you also need 2 more plates as controls.
What should these 2 control plates be transfected with, and should you add growth factors to 0, 1 or both of these plates?
2.3 There are 2 serines in the 100 amino acid SAP1 activation domain (SAP1 AD) that your labmate mutates to alanine in one construct, or aspartic acid in another. These are then fused to the Gal4 DBD to make Gal4-SAP1 AD Ser->Ala and Gal4-SAP1 AD Ser->Asp respectively. You transfect 2 plates of fibroblasts with Gal4-SAP1 AD, 2 plates with Gal4-SAP1 AD Ser->Ala and 2 plates with Gal4-SAP1 AD Ser->Asp and add growth factors to one of the 2 plates for each Gal4 fusion. You finish the experiment but become confused which plates you transfected with Gal4-SAP1 AD Ser->Ala and to which you added Gal4-SAP1 AD Ser->Asp. However, you are sure which plates you added growth factors to and which plate had the normal Gal4-SAP1 AD.
Which of the expression vectors (m1 or m2) is Gal4-SAP1 AD Ser->Ala and which is Gal4-SAP1 AD Ser->Asp? Briefly explain your answer.
Q3. ChIP
3.1 What is ChIP an abbreviation for?
3.2 Briefly explain the main differences between ChIP and a band shift (gel shift) assay.
3.3 R represses transcription of gene Z via site Z1 in the Z regulatory region. Using ChIP, you find that R immuno-precipitates on the Z1 site in mammalian cells in culture. However, you cannot obtain a gel shift with purified R protein on the Z1 site even though you try all kinds of variations in the reaction buffer to help R bind DNA (e.g. adding zinc etc.). You are concerned, but the head of your lab tells you that you are doing the gel shift correctly, and that your data are actually informative about how R represses transcription. What model could explain your data?
4. One more gel shift question
You are using a 150 bp labelled DNA as a gel shift probe and you add increasing concentrations of a DNA-binding protein (DB1) from lanes 2 to 4. You see the following pattern on the autoradiograph of the gel shift.
You show this to 2 labmates: one suggests that the pattern is because DB1 can form tetramers on a single site, while the other labmate suggests that the pattern is because there are two different binding sites for DB1 in the 150bp DNA fragment.
You decide to test the idea by cutting the 150bp DNA probe into two equal-size 75bp pieces which you use as probes (Probe 1 and Probe 2) .
4.1 Draw what you would see if DB1 can form. tetramers on a single site
4.2 Draw what you would see if there is one DB1 binding site on each 75bp probe