Lecture 5: RNA Structure and Transcription
After this lecture, you should be able to answer the following questions:
1. What are introns? Exons? What are nucleosomes? Describe nucleosomes to your study partner. What property of histone proteins enables them to interact with DNA? How can histones be modified? What are the names of two enzymes that acetylate histone proteins? What is the net effect of histone acetylation? What about enzymes that de-acetylate histone proteins? What are they called? What is the net effect of histone de-acetylation on transcription?
2. What are the bases found in RNA? Are thymines present in RNA?
3. What are the different types of prokaryotic RNA? Decribe the structure of prokaryotic mRNA. What is a Shine-Delgarno sequence?
4. Describe promoter structure in prokaryotes. What is the -10 region? The -30 region?
5. Describe the prokaryotic RNA polymerase (the core polymerase) and its role in transcription. What do each of the subunits do? Which of the subunits of this multisubunit protein is the target of the antibiotic rifampicin? What is the role of the sigma factor (recall that core polymerase + sigma factor = ‘holoenzyme.”)
6. What is the lac operon? Explain why it represents a good example of transcriptional regulation in response to metabolic needs of bacteria.
7. What are the different types of eukaryotic RNA? How many eukaryotic RNA polymerase are there? Which eukarotic RNA polymerase is responsible for the synthesis of hnRNA/mRNA? Which of the eukaryotic RNA polymerases is most sensitive to alpha-amanitin?
8. Describe the structure of eukaryotic mRNA. How does it differ from the structure of prokaryotic mRNA?
9. Discuss the types of transcription and promoter elements in eukaryotic DNA.
10. What are the names and functions of the proteins that regulate mRNA synthesis in eukaryotes?
11. What is a transcription factor? Give an example of a basic-region leucine zipper (bZIP) transcription factor. Describe how binding of AP1 to DNA activates transcription.
Kit Corner: 1. Why is fluorescently-tagged Annexin V a good tool to use in the study of apoptosis? where does phosphatidylserine come into the picture?
2. HDAC inhibitors promote the continued acetylation of histones bu blocking histone deacetylase. What does that mean for DNA? Loose nucleosomes or tight ones?
3. Syber Green reagent is used in RT-PCR procedures such as the Shine Polo One Step RT-PCR kit. Why does the use of Syber Green in this assay eliminate the need for electrophoresis?
4. What effect would an inhibitor of P-gp have upon to the levels of NADH in the P-gp Drug Interaction Assay Kit (www.spibio.com)?
Lecture 4: DNA Replication, Mutation, and Repair
Students: Our class is cancelled. But I have emailed you the slide set. Please read it and answer the questions below. Note that our next meeting is February 24th!
Try to get through your Background and Significance by then. On the 24th, I will explain the Preliminary Studies section. Good luck!
After reading this slide set, you should be able to answer the following:
1. Describe DNA replication in E. coli. Where does replication begin? Anywhere on the chromosome or at a specific site? How many such sites are present on the E. coli chromosome? How many such sites are present in human chromosomes? What are the proteins involved in DNA replication in E. coli (name the protein and its function)? What are "replication bubbles?" What are "replication forks?" What do we mean when we say that a DNA polymerase has proofreading activity?
2. Describe the different types of gene mutations.
3. What is the AMES test? what is the principle behind the test?
4. What is mismatch repair? What are the different types of mismatch repair? What are the enzymes involved in these two types of mismatch repair?
Lecture 3: Chromosomes
After this lecture, you should be able to answer the following:
[NOT COVERED IN LECTURE]
1. What are the structural features of chromosomes? What are monads? dyads? when do we see monads? at what point during the cell cycle do we see dyads? How many pairs of chromosomes are in the human genome?
2. What is the cell cycle? What are the different phases of the cell cycle?
3. Explain the process of mitosis. What are the different stages of mitosis? What happens during those stages?
4. What is meiosis? What are the stages of meiosis?
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6. What is meiotic recombination? How does it contribute to genetic diversity?
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7. What did Griffith's experiment demonstrate regarding transformation? How did Avery, MacLeod and MacCarty's experiment help clarify that DNA was the hereditary material? Explain the Hershey-Chase experiment. What did that experiment demonstrate?
8. Discuss the structure of DNA from a biochemical perspective. What are the chemical components that make up DNA? What is Chargaff's law? What is the role of hydrogen bonding? How can hydrogen bonds be broken? Which base pairs share the strongest H-bonds?
9. Diagram the kinetics of DNA renaturation for human genomic DNA. Explain the three "bumps on the curve."
10. What is repetitive DNA? What are two types of repetitive DNA? What are Alu sequences?
11. What is the size of the human genome? Approximately how many genes make up our genome?
KIT CORNER:
p38 MAP Kinase Assay:
1. What type of assay is this? What kind of instrument do you need to perform this assay? Briefly describe the theory underlying this kit.
20S Proteasome Kit:
1. What type of assay is this? What is the 20S proteasome? What kind of instrument do you need to perform this assay? Briefly describe the theory underlying this kit.
DNA Damage Quantification Kit:
1. Why is an aldehyde reactive probe used in this kit to detect abasic sites within DNA? (Note: An AP site is also known as a apurinic/apyrimidinic site which is also known as an abasic site). What kind of instrument do you need to perform this assay?
Glutathione Quantification Kit
1. What kind of instrument do you need to perform this assay? Why is glutathione important? What is the difference between reduced (GSH) and oxidized (GSSG) forms? What is DTNB?
