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Updated SQ 21-23.docx

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University of Waterloo
BIOL 308
Dragana Miskovic

STUDY QUESTIONS: LEC 21-23 1. What are the major differences between prokaryotic and eukaryotic transcription? Transcription and translation in eukaryotes occurs in separate compartments. Eukaryote pre-MRNAs are subject to extensive post-transcriptional modification processing. Chromatin structure in eukaryotes limits accessibility and transcription is highly regulated. Eukaryotic RNAP does not recognize binding sites and needs general TFs to help. Eukaryotic transcription has 3 different RNAPs, each with different roles. Eukaryotic transcription has no operons. 2. Which hypothesis regarding eukaryotic RNAPs was proven with a-amanitin and actinomycin D (be specific)? The hypothesis that different RNA polymerases are involved in transcribing certain genes but not others and had different roles in transcription was proven biochemical analysis with the compounds a-amanitin (decreases the activity of RNA polymerase II) and actinomycin D (decreases the activity of RNA polymerase I). The compounds were injected in mouse cell nuclei and then the transcripts produced were subject to gel electrophoresis. Certain genes were proven to be transcribed while others were not. Thus, their transcriptional levels were dependent upon which polymerase was inhibited. 3. Which genes are transcribed by RNAP I? RNAP II? RNAP III? RNA polymerase I: transcribes rRNA (28S, 18S and 5.8S rRNA) RNA polymerase II: transcribes mRNA, snRNAs and miRNA RNA polymerase III: transcribes tRNA, 5S rRNA, U6 snRNA 4. What does CTD stand for? Explain the role of CTD tail in eukaryotic gene expression? CTD stands for carboxy terminal domain. It is a consensus sequence which is unique to RNA polymerase II. The dephosphorylated (II a) form is required for RNAPII binding to the PIC. The phosphorylated form (II o) is required for elongation. Also roles in splicing, methylation, and polyadenylation. 5. How would you define enhancers? What are their characteristics? What is the difference(s) between enhancer and upstream control element? Enhancers are genetic elements to which activators can bind and increase the ability of RNAP to bind the promoter – they can be found anywhere. They are located at a considerable distance (up to 50kb) from the start point of transcription. Sometimes the same element can be both an enhancer and silencer depending on the bound protein. Upstream control elements are only found upstream of the promoter and are sites for regulatory proteins to bind and influence transcription. 6. Explain the use of reporter genes for estimation of promoter strength. Reporter genes are coding units whose product (either enzymes or a fluorescent protein) is easily assayed when the genes are expressed. When placed under control of a promoter, the strength of the promoter can be measured by measuring the level of product production (GFP, beta-galactosidase, luciferase are examples). The strength is proportional to the amount of gene product produced. 7. Explain briefly 5’ deletion series. What kind of information do they reveal? 5’ deletion is done by mutating or deleting parts of the promoter starting from the 5’ end. The level of transcription of the reporter gene is compared to that of the control (original promoter with no deletions). By comparing the two levels, the function of the area deleted can be deduced (if the transcript level decreases, the area deleted is important for one or more steps of the transcription process, however, if the level increases, the area deleted is an inhibitory region). 8. Explain the modular nature of RNAP II promoters. RNAP II promoters are modular in that they are organized on the principle of “mix and match”, and there is no one element that is essential for (or common to) all promoters. In any individual promoter, elements may differ in number, location, and orientation, and thus different combinations can achieve different levels of expression. Expression of one gene can be induced or increased or decreased or shut-off under different conditions due to the modularity of gene's promoter. 9. Draw a diagram of RNAP II promoter (show all types of elements that we have mentioned in class). 10. Explain the tissue (cell type) specificity of eukaryotic cis elements. Since DNA content is the same in every cell, the cis elements and promoters are similar as well. However, tissue specificity is due to either the presence or absence of a particular regulatory/binding protein known as transcription factors. These TFs dictate tissue specificity of cis elements at the level of initiation of transcription. 11. Knowing that different genes may have the same promoter and enhancer elements and that different transcription factors contain the same structural features, how would you explain transcriptional specificity? Although different TF contain the same structural features, the way they are used and what they bind to (modular nature of promoters) allow for transcriptional specificity. It is the combinatorial control using TF that controls how TF brings out the effect. Also a response has its corresponding cis element and similar responses have similar cis elements. E.g. the heat shock proteins are transcribed in response to high temperature as well as heavy metals. Heat shock response elements (HRE) and metal responsive elements (MRE) are the TFs that control the transcription of the gene. 12. If you know the binding site for certain transcription factor (TF), outline experiments you would use to purify this TF and to assay its activity. To purify: The gene would be shredded into pieces and would go through beads containing TF binding sites. This would leave TF bound to the beads and the other parts are flushed down. The TF would be eluted with high salt water. (Ion exchange chromatography, DNA-affinity chromatography and specific DNA-affinity chromatography) To assay its activity, two plasmids would be made; one with TF gene and the other with binding site for TF and reporter gene. When they are inserted into a host, TF gene would be transcribed so that its product (protein) may bind to the TF binding site, causing the reporter gene to be transcribed. The product of the reporter gene can then be measured. 13. Distinguish between the function of promoters and enhancers in transcriptional regula
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