PSwk8 - Problem Set wk 8 MCB 110, Spring ‘11 1....

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Problem Set wk 8 MCB 110, Spring ‘11 1. True or False ____ High sequence conservation is generally an indication of functional significance ____ Insulator proteins establish boundaries around a promoter and its enhancers so that other promoter/enhancer elements are not impacted ____ Transcription factors with leucine zipper motifs can only form homodimers ____ Histone tails are important for formation of tightly wrapped DNA around the histone octamer ____ Acetlyation of histone N ­terminal tails always leads to gene repression ____ Methylation of histone tails always results in gene activation ____ All the nucleosomes at active genes are evicted by remodeler complexes both at the promoter and along the gene sequence so that polymerase can travel along the DNA unhindered ____ Euchromatin is a condensed structural state of chromatin ____ ATP is required to slide a nucleosome ____ HDAC and HAT proteins perform the same functions ____ NELF and DSIF need to be phosphorylated for Pol II elongation ____ TAT is one of the host proteins that HIV virus uses 2. Chromatin immunoprecipitation is a valuable tool to determine which sequence or region of DNA a particular DNA ­binding protein binds. However it requires some prior knowledge about the sequence, either from DNaseI footpringting or gel shift. What kind of technique could you use that requires no prior knowledge and also gives you a comprehensive and unbiased look at which sequences are bound? What would your experimental (positive and negative) controls be? 3. You’ve identified (cloned) a new protein that is localized in the nucleus. a) How can you determine if it’s a transcription factor by looking at its sequence? b) You did a ChIP ­seq with an antibody against this protein and another one with an antibody against H3 lysine 27 methylation. The sequences you obtained from the two samples overlap nearly perfectly, what does this say about your transcription factor? X - Human mtakmettfy ddalnasflp sesgpygysn pkilkqsmtl nladpvgslk phlraknsdl ltspdmgllk laspelerli iqssnghitt tptptqflcl knvtdeqegf aegfvralae lhsqntlpsv tsaaqpvnga g4. Your lab is studying a haslhseppvy anlsnfnpga lssgggapsy gaaglafpaq pqqqqqpphh mvapavasv aggsgsggfs uman oncoprotein, X, that is a component of the mammalian rlqalkeepq tvpempgetp plspidmeSQ ERIKAERKRMproliferation lpqqmpvqhp transcription factor AP1 and involved in activation of RNRIAASKCR genes. You know the identity and the sequence o kvmnhvnsgc ith the following KRKleriarl eekvktlkaq nselastanm lreqvaqlkq f the protein wqlmltqqlqt f amino acid sequences: Deletion mutant # 1 = the blue bold italics (domain 1) of the protein sequence was mtakmettfy ddalnasflp sesgpygysn pkilkqsmtl nladpvgslk phlraknsdl deleted ltspdmgllk laspelerli iqssnghitt tptptqflcl knvtdeqegf aegfvralae lhsqntlpsv Deletion mutant # 2 = the pink bold caps (domain 2) of the protein sequence was tsaaqpvnga gmvapavasv aggsgsggfs aslhseppvy anlsnfnpga lssgggapsy deleted gaaglafpaq pqqqqqpphh lpqqmpvqhp rlqalkeepq tvpempgetp plspidmeSQ Deletion mutant # 3 = the green bold underlined (domain 3) of the protein sequence ERIKAERKRM RNRIAASKCR KRKleriarl eekvktlkaq nselastanm lreqvaqlkq was deleted kvmnhvnsgc qlmltqqlqt f You make three deletion mutants: 1 ­the blue domain is deleted; 2 ­ the purple region To test the transcriptional effect of these is deleted, and 3 ­the green region is deleted. deletions, your lab did a series of experiments. First you did an in vitro transcription assay using a wild-type promoter with the transcriptional e the reconstituted general transcription factors. Then you To test the AP1 site and all ffects of these mutants, you did an in vitro transcription did a g a shift assay using the wild-type promoter with the AP1 binding site, assay elnd found that the wildtype protein was the most active with all three and last you did a size exclusion chromatography gel shift assay and got the following mutants being non ­active. Then you did aexperiment to test the difference between mutant results: 2 and 3. Here is your data: a) what type of transcription factor is this? (think back on the three different motifs we’ve learned a) What type of transcription factor is this, and what purpose do each of these domains that you deleted have, how do you know? b) What is the purpose/function of each of the three domains that you deleted in your mutants? How do you know? c) Would you hypothesize that this transcription factor works as a monomer, a dimer, a trimer, or a tetramer? Why? What type of DNA sequence motifs would you expect its binding site to have? 5. While many transcription factors function by affecting initiation of transcription, some transcription regulators function by affecting elongation, partially supported by ChIP ­seq where you see Pol II localized to genes that are actually untranscribed. a) Without doing an RNA transcript analysis, how can you use ChIP to determine which genes have Pol II sitting at the promoter and which ones have Pol II actively transcribing. What features of the polymerase would you exploit for this purpose? b) What should you include as negative and positive controls? 6. What is the TAR RNA? What is it transcribed from and how does it stimulate transcription of the HIV gene? 7. List at least two substrates that P ­TEFb phosphorylates 8. What are the three different ways that nucleosomes or effects on nucleosomes can regulate transcription? 9. What are the advantages for many transcription factors to form heterodimers? 10. The HIV protein Tat is required for HIV propagation. What is the function of Tat? 11. Cyclic AMP serves as a second messenger for intracellular signaling. Describe how cAMP can lead to transcription activation. Start your description from binding of a hormone to a receptor. 12. Regulators are regulated in various different manners. List all of the mechanisms you’ve learned from class. 13. What mechanism of regulation is used to regulate NF ­kB transcription factor? 14. What feature of Nuclear Receptor ligands is key for their function? 15. Imagine you are working with a Nuclear Receptor protein called Rambo. Your PI gives you three different candidate ligands for this Nuclear Receptor. Describe an experiment that would allow you to determine which of these ligands is the real ligand for Rambo. Explain what the data would show for the real ligand and for the other ligands. ...
View Full Document

This document was uploaded on 09/12/2011.

Ask a homework question - tutors are online