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Department
Biology
Course
BIOL 308
Professor
Dragana Miskovic
Semester
Summer

Description
1. What are the major differences between prokaryotic and eukaryotic transcription? A. Eukaryotic transcription is carried out by 3 RNAP compared to 1 in Prokaryoutes. B. RNAPs of eukaryotes cannot initiate transcription without the help of general transcription factors (needed for binding to DNA). C. The primary transcript in Eukaryotes contains introns which have to be spliced to produce the mature mRNA. The process of maturation also includes adding a 5’ Cap and a 3’ poly A tail. D. The chromatin structure in eukaryotes limits access of the enzymes to the genes. This helps regulate the process of transcription. (0.01% transcriped at any given moment, approximately 3 genes). N.B: RNAP IV plays a role in gene silencing. In plants, it transcripes specific RNAs that are involved in gene silencing. 2. Which hypothesis regarding eukaryotic RNAPs was proven with α-amanitin and actinomycin D (be specific)? Hypothesis: There are 3 RNAPs in Eukaryotes (I, II and III). It also proved they played different roles in transcription. ά-amanitin is an inhibitor of RNAP II. It’s a lipid soluble molecule (can cross the membrane) found in mushrooms (Death cap and Destroying angel). It can be used to identify the type of RNAP in action since RNAP I is insensitive to it, RNP II is highly sensitive and RNAP III is only slightly sensitive. Actinomycin D is an antibiotic produced by Streptomyces that intercalates into GC rich regions of DNA inhibiting their transcription. (RNAP can no longer bind to them) RNAP I is the most sensitive to it. It can be used in chemotherapy. 3. Which genes are transcribed by RNAP I? RNAP II? RNAP III? (details required for proceeding lectures) a. RNAP I transcripes large rRNA precursors such as 28S, 18S, and 5.8S b. RNAP II transcripes hnRNA which undergoes processing to produce mRNA (inhibition of RNAP II by ά-amanitin is fatal). c. RNAP III transcripes 5S rRNA and tRNA mainly. Hint: RNAPI = rRNA. RNAPII = mRNA RNAPIII = tRNA. 4. What does CTD stand for? Explain the role of CTD tail in eukaryotic gene expression? CTD stands for Carboxy-Terminal Domain. The large subunit of RNAP II (only) contains the CTD which can be in a: a. Un-Phosphorylated (II a): involved in binding the RNAP to the promoter. b. Phosphorylated form (II o): involved in elongation as well as splicing, addition of the 5’ CAP and the 3’ Poly-A tail (i.e. all post-transcriptional modifications) and termination. 5. How would you define enhancers? What are their characteristics? What is the difference(s) between enhancer and upstream control element? a. An enhancer is a sequence of DNA (cis element) that binds to transcription factors like activators or silencers to influence the rate of transcription. They can have multiple binding sites for different transcription factors and are independent of the orientation and location (i.e. upstream, downstream, intron, exon). b. Upstream elements are only found upstream and in proximity to the promoter. N.B: RNAP are made up of 8-14 subunits. 5 of which are common to all three RNAPs as they perform similar functions. RNAPs of Eukaryotes require general TFs to initiate transcription. CTD tail: Stretch of 7AA that are repeated multiple times (>10 but number is specific to the organism). 5 of the 7AA have an OH group and therefore, the tail can be phosphorylated (into OP) and is hydrophilic. Refer to Q4 for functions. Eukaryotes have NO operons. Instantaneous mRNA level is controlled by transcription rate as well as degradation rates. 6. Explain the use of reporter genes for estimation of promoter strength. Reporter genes are genes whose products are easily assayed. They are incorporated after the promoter whose activity is to be measured. When the promoter is activated and gene transcription starts, the reporter gene is also transcriped and the level of formation of its product is proportional to the strength of the promoter. 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). It may be used to locate promoter, enhancer, repressor and silencer. E.g.: Lac Z gene produces β-galactosidase which hydrolyses X-Gal (yellow) into a colorless galactose and blue 4-chloro-3-bromo-indigo. (yellow>>blue) 8. Explain the modular nature of RNAP II promoters. RNAPII promoters are composed of core promoter and enhancer/silencer (cis elements). There are variety of different core promoters and elements and none is vital for all promoters. Depending on the situation and desired outcome, a combination of a promoter and cis element(s) are brought together. Cis elements include TATA box, initiator, and CG sequence. N.B: Promoter is a sequence that must be located in the vicinity of the start point and are required for initiation of transcription. Most class II promoters contain a core element as well as a regulatory element. Core promoter: the minimal set of elements required for accurate in vitro transcription initiation by RNAP II. They are necessary for recruitment, binding and proper positioning of RNAPII. E.g. 1. TATA box (position at ~ -30): It binds TBP which is involved in initiation of transcription for RNAP I and III as well as TATA less promoters. 2. Initiator (Inr; on the transcription start site). 3. Downstream element (DPE; downstream). 4. TF II B recognition element (BRE) Regulatory elements influence transcription rate through binding of regulatory proteins. They influence the positioning of the RNAP. However, they are sometimes necessary for initiation of transcription. They are classified in respect to the distance from a core promoter and activity (+ vs. – effect on transcription) of the element Upstream elements which are close to the promoter (GC box, CCAAT box and promoter- proximal elements). Enhancers and silencers (everywhere, orientation is irrelevant) Also boundary elements and insulators For details, refer to Lecture 21 notes. 9. Draw a diagram of RNAP II promoter (show all types of elements that we have mentioned in class). Enchancers: closely arranged elements that bind transcription factors, can be everywhere, orientation independent. Upstream elements: dispersed element, close to promoter, influence transcription. Enhancers/silencer Regulatory elements s Upstream elements Core promoter element (e.g. TATA box) 10.Explain the tissue (cell type) specificity of eukaryotic cis elements. DNA content of every cell is the same, therefore, cis elements as well as the promoters are the same. However, Transcription factors are specific to every cell and control the function of the cis elements and therefore, the transcription of genes. Therefore, they are responsible for the specificity of the cells. 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 transcriped 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. N.B: genes that respond to multiple conditions have multiple cis elements. 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. The gene would be shredded into pieces and would go through beads containing TF
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