Answer Key: Making mRNA (Transcription)

BIOL230W Week 3 – Making mRNA (Transcription) We are continuing our journey following the flow of information in a cell. Last week we focused on DNA, it’s structure, function and how it’s stored in a cell. This week will look at how information is organized in the DNA (i.e., genes vs. promoters vs. introns) and how cells “express” this information to form gene products like proteins. Day 1 Learning Objectives 1. Define "genome" and how genomes may vary between organisms. 2. Describe the regulatory and structural components of a eukaryotic genome. 3. Explain the connection between multicellularity and genome size. 4. Define "differential gene expression". 5. Compare and contrast the major proteins of the transcription initiation complex (TIC): basal transcription factors, co-activators, activators and repressors 6. Explain the relationship of the DNA-binding proteins to target DNA sequences (enhances, silencer, TATA box, etc.) 7. Analyze how the TIC forms and what would result if any protein or DNA sequence is altered. 8. Understand how RNA polymerase is positioned at the start of transcription. 9. Explain how the transcription rate can be altered through the formation of the TIC Small Group Activity: Genome Size Across Organisms 1. How do you define a genome? All of the genetic information in a cell Look overFigure 1 (below) with your group. Make at least three observations about the data. Hint, compare the slopes of the 4 different categories of organisms. Then answer the following questions. OBSERVATIONS Archaea, Bacteria, and Viruses all have linear progression of increasing from protein coding genes to genome size. Amount of protein coding genes in eukaryotes tapers off, so something other than protein coding genes is being added to increase genome size. Viruses contain the fewest amount of protein coding genes as well as the smallest genome size. Figure 1 – Comparison of total genome size vs. protein coding regions in four categories of organisms 2. Which group of organism has the largest genomes? Eukaryotes 1 BIOL230W Week 3 – Making mRNA (Transcription) 3. Do eukaryotic genomes have linear relationship between genome size and genomes (i.e., as the genome increases, the number of genes increase at the same rate)? What does your answer mean? No, it begins to plateau. This means that something other than protein coding genes is being added to increase the genome size. Now think about the cells in your body… 4. What makes a neuronal cell different from a muscle cell? They have the same genetic makeup, but they express genes differently (differential gene expression). Some expressed at higher and lower levels depending on the type of cell they are in. Small Group Activity: Initiation of Transcription Look overFigure 2 below. Thenread the legend and answer the questions that follow. Nucleosome Activator RNA Polymerase Transcription Factors Coactivator Figure 2. The eukaryotic transition initiation complex. Basal transcription factors include many complexes: TFIIA (3 polypeptides), TFIIB (1 polypeptide), TFIID (12 polypeptides – including TAFS and TBP), TFIIE (2 polypeptides), TFIIF (2 polypeptides), and TFIIH (9 polypeptides). TFIIA, TFIIB, and TFIID recognize the promoter-proximal DNA elements including the TATA box, the BRE (TFIIB recognition element), and the initiator (INR) element. During initiation, TFIIF binds to Pol II (RNA polymerase) and helps to recruit the enzyme to the start of the gene. TFIIE binds and helps recruit TFIIH to the promoter region. TFIIH separates the strands of DNA allowing Pol II access to the template strand. ISWI (imitation switch) and HATs (histone acetyl transferases) work to remodel chromatin structure. CRSP/MED enhances initiation by stabilizing interactions between enhancer-bound activators and the basal transcription factors (see arrow). TBP - TATA binding peptide, RE - response element (silencers and repressors) -35 and -44 are CFTR specific REs that regulate expression in different tissue types; TAF - TBP-associating factor. From Regulatory diversity among metazoan co-activator complexes. Taatjes et al., Nature Reviews Molecular Cell Biology 5, 403-410 (May 2004) 5. Label/highlightRNA Polymerase,activator, coactivator, nucleosome,and basal transcription factors. 2 BIOL230W Week 3 – Making mRNA (Transcription) 6. Which transcription factor (i.e., TFIIA, B, D, etc.) binds to the TATA-box? TBP, or TATA-binding protein. 7. Why is TATA-box binding important regarding RNA polymerase? Helps transcription factors position the polymerase in the right spot. 8. What is the TATA box? Sequence of DNA, up from promoters. 9. What type of macromolecule is IRF-1 and NRF-2? What is their role in gene expression? Activator proteins that bind to enhancer regions on DNA. They increase the rate of transcription. 10. Would transcription occur if there was a loss-of function mutation in CRSP/MED? Why or why not? Why or why not? Yes, transcription factors are the only thing necessary. Therefore, transcription would still occur just at a lower rate. Day 2 Learning Objectives 1. Identify the DNA ends (5' and 3') involved in transcription and how those ends are used to describe the direction of transcription. 2. Compare and contrast terms: coding, template, sense and anti-sense. 3. Describe the three processes that occur during pre-mRNA to mature mRNA formation. 4. Compare and contrast the RNA and protein components of snRNPs (and their roles in function of a splicesome). 5. Explain alternative splicing and analyze how different proteins may arise from the same mRNA. 6. Relate the different cellular locations with function (i.e., transcription = nucleus, pre-mRNA processing = nucleus, ect.). Mini-Lecture: Making mRNA - Elongation 1. In which direction is the template strand read ? 3’ to 5’ 2. Which carbon on the nucleotide sugar contains the OH? The 3’ carbon 3. Which carbon on the nucleotide sugar ribose contains the 4? PO The 5’ carbon 4. The phosphate of the incoming ribonucleotide is added to the oxygen on which carbon? The 3’ carbon 3 BIOL230W Week 3 – Making mRNA (Transcription) 5. In which direction is the RNA molecule made by the RNA polymerase? 5’ to 3’ Small Group Activity: Maggie’s Primary Transcript A researcher has a patient, Maggie, with cystic fibrosis. The researcher has sequenced the CFTR gene from the patient’s DNA and from the DNA of an individual without CF. Use what you know about transcription to determine the sequence ofprimary the transcript (pre-mRNA) for both the patient with CF and the individual without based on the DNA sequences shown below. We will come back to add in the mature mRNA in a moment. You only need to transcribe the region in the purple (shaded) box. 6. Primary Transcript (pre-mRNA): AACAGUUGCAG…AUCAUCUUUGGU 7. Mature mRNA: AACCAG…AUCAUCUUUGGU 8. Primary Transcript (pre-mRNA): AACAGUUGCAG…AUCAUCGGU 9. Mature mRNA: AACCAG…AUCAUCGGU Summary 10. What is the difference between Maggie’s DNA and the individual without CF? There are some nucleotides missing (3 missing, TTT) 11. What is the difference between Maggie’s mature mRNA and the individual without CF? There are some nucleotides missing (3 missing, UUU) 4 BIOL230W Week 3 – Making mRNA (Transcription) Day 3 Learning Objectives: 1. Compare and contrast mutation and epigenetic regulation. 2. Describe 3 types of single nucleotide mutations. 3. Describe the general role of epigenetics in gene expression. 4. Compare and contrast DNA methylation and histone modification mechanisms of epigenetics from Week 3 scientific literature reading. 5. Apply the concept of methylation sensitive restriction enzymes to determine methylation status Mini-Lecture: Recap of Differential Gene Expression Figure 3: Cap analysis gene expression (CAGE) isolates small fragments from the 5' ends of the RNA and then, reverse-transcribes them to DNA, PCR amplifies and sequences them. The number of times a sequence is transcribed gets tallied and gives a quantitative measure of expression of this gene in a given sample (tissue). 1. What is this graph measuring? X-Axis= Different tissues Y-Axis= Scaled tags (per million) 2. In which six tissues are CFTR most highly expressed? Small intestine, Colon, Pancreas, Gallbladder, Appendix, and Lung 3. How does this relate to the CF phenotype? Each symptom is related to these tissues, due to the increased thickness of mucus (ex: constipation caused by mucus being too thick in small intestine) Small Group Discussion – Changes in Phenotype 4. What is a genetic mutation? 5 BIOL230W Week 3 – Making mRNA (Transcription) A change to nucleic acid sequence of an organism’s genome, can also apply to viruses and extrachromosomal DNA. 5. What can be the cause of mutations? Brainstorm a few ideas. Genetics (familial), environment, mistakes in transcription/replication 6. Why does a mutation change your phenotype? Genotypes effect the expression of a phenotype 7. Why might one person have cystic fibrosis and one person does not? Cystic Fibrosis is recessive, so someone can carry one CF allele and not show the phenotype. Therefore, if someone has a mutation in the CFTR gene, they will need the mutation to be present in both alleles to express the phenotype. 8. How can the phenotypes of identical twins be different (DNA sequences are the same)? Environment can differ, so they may have the same phenotype but express it in slightly different ways. Small Group Activity: Identifying Methylated DNA Restriction endonucleases are DNA modifying enzymes that act at specific DNA sequences to break the phosphodiester bonds holding the DNA polymer together. Cleaving the phosphodiester bond on both DNA strands will break the DNA into two pieces. These pieces can then be separated and analyzed via agarose gel electrophoresis. Some restriction endonucleases are sensitive to methylation of DNA and can be used to determine if DNA has been methylated. HpaII is such an endonuclease. HpaII normally nd cuts at the sequence CCGG, but if the cytosine 2 is methylated it will not cut. Figure 8. (A) The horizontal line represents a stretch of DNA that contains three CCGG HpaII sites, indicated with orange arrowheads. The numbers below the line and between the HpaII sites indicate the distance in base-pairs between the cut sites. The numbers on the left and right ends of the stretch of DNA indicate more than 2000 base-pairs before the next HpaII site. (B) Representation of an agarose 6 BIOL230W Week 3 – Making mRNA (Transcription) gel after electrophoresis. Two samples of DNA were treated with HpaII enzyme for 2 hours at 37˚ C and then run for 30 min at 100 volts in TAE buffer. DNA fragments larger than 1500 bp are not shown. 9. Which of the DNA samples has been methylated at one or more HpaII sites? Sample 2 10. Which site (s) was (were) methylated in DNA sample 1 vs. DNA sample 2? Site 2 on sample 1 Optional End of Week 3 Summary Drawing – For your own practice (will not be graded) Summarize what you have learned this week with a drawing in the space below. Draw and label a gene being transcribed into mature mRNA. Include the following: The DNA molecule (both strands), coding strand (sense), template strand (antisense), RNA polymerase, basal TFs, silencer, enhancer, activator, repressor, splice site, pre-mRNA, mature mRNA 7