Making mRNA (Transcription)

BIOL230W Week 3 – Making mRNA (Transcription) Mariana Kaufman 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? The full set of genetic material in the cell or an organism Look over Figure 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 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? Eukaryota have the largest genomes. Mammels have 4 times more the genes than yeast. 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? They do not have a linear relationship, As genome size increases we can see a plateau. This is what allows for differential gene expressions. Eukaroyes receive more DNA but less protein coding genes—> less linear Now think about the cells in your body… 4. What makes a neuronal cell different from a muscle cell? Even though all cells have the same DNA, not all cells express the same gene at the same levels—> leads to unique cells types Differential gene expression Mini-Lecture: Transcription Factors Small Group Activity: Initiation of Transcription Look over Figure 2 below. Then read the legend and answer the questions that follow. Nucleosome ↳ activator coactivator e Transcription RNA Polymerase - Figure 2. The eukaryotic transcription 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).(bind to promoter) 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 (enhancers are shown) -35 and -44(enhancers) 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/highlight RNA Polymerase, activator, coactivator(binds to activator and basal transcription factors), nucleosome and basal transcription factors. 6. Which transcription factor (i.e., TFIIA, B, D, ect.) binds to the TATA-box? The TBP binds to the TATA box recruits the basal transcription factors TATA binding protein can slightly change confirmation to form stability to bring in RNA polymerase 7. Why is TATA-box binding important regarding RNA polymerase? TBP binding to the TATA box helps initiate basal transcription factors for transcription It helps regulate expression in different tissue types So it can start reading the DNA transcribed at a constant rate 8. What is the TATA box? Sequence of DNA in the promotor AT rich region which is bound with a TATA bounding protein to start the process 9. What type of macromolecule is IRF-1 and NRF-2? What is their role in gene expression? IRF-1 and NRF-2 act as activators 10. Would transcription occur if there was a loss-of function mutation in CRSP/MED? Why or why not? You still could but at a lower rate RNA polymerase cannot be recruited decrease in gene expression—> Decrease in failure to pump ions-failure to thin mucus 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 vs. template strands 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’-5’ 2. Which carbon on the nucleotide sugar contains the OH? the 3’ carbon of the deoxyribose sugar 3. Which carbon on the nucleotide sugar contains the phosphate group (PO4)? the 5’ carbon is where the phosphate group is attached 4. The phosphate of the incoming ribonucleotide is added to the oxygen on which carbon? 3’carbon 5. In which direction is the RNA molecule made by the RNA polymerase? 5’—> 3” Small Group Activity: 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 of the primary 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. Note: the promoter region is labeled and boxed, the transcriptional start site is labeled, and the intron is the underlined sequence. We will only be examining the transcription of region in the purple (shaded) box. 6. Primary Transcript (pre-mRNA): 5” AACAGUUGAS…AUCAUCUUUGGU3” * 7. Mature mRNA: 5’ CAP ACGCA…AUCAUCUUUGGU(AAAA) 8. Primary Transcript (pre-mRNA): 5’AACAGUUGCAG….AUCAUCGGU3' * 9. Mature mRNA: 5’ cap AACGCAGCAG…AUCAUCGGU3’ (AAAA) Summary 10. What is different between Maggie’s DNA and the individual without CF? we see that maggies DNA is missing the TTT near the 3’ end 11. What is different between Maggie’s RNA and the individual without CF? Maggies DNA is missing the uuu near the 3’ end Day 3 Learning Objectives: 1. Describe the difference between a mutation vs. 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 Small Group Activity: Recap of Differential Gene Expression 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= The x axis is measuring different tissues types Y-Axis= the y axis is measuring the number of times that it is transcribed ( scale tags) 2. In which six tissues are CFTR most highly expressed? The six tissues that are most highly expressed are the small intestine, the colon, the pancreas, the gallbladder, the appendix and the lung. 3. How does this relate to the CF phenotype? We can see that the issues are mainly in the respiratory tract where CF is usually effecting Mini-Lecture – Changes to Phenotype 4. What is a genetic mutation? A gene mutation is a chromosomal change to a single nucleotide 5. How can a mutation change your phenotype? Deletion, insertion, relocation 6. What can be the cause of mutations? Mutations can be caused by your environment, genetics and/or mistakes in transcription/replication. 7. How can the phenotypes of identical twins be different (DNA sequences are the same)? A genotype can have multiple phenotypes Mini-lecture: Mutations and Epigenetics Small Group Discussion: 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 cuts at the sequence CCGG, but if the 2nd cytosine 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 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. 8. Which of the DNA samples has been methylated at one or more HpaII sites? Both A and B’s DNA samples have been methylated or more Hpall sites 9. Which site (s) was (were) methylated in DNA sample 1 vs. DNA sample 2? In the sample A, we see there were 3 sites methylated and in sample B 2 sites were methylated which is shown by the gel electrophoresis.