Molecular Biology (BIOL 4320) Exam #1 March 12, 2002

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1 Molecular Biology (BIOL 4320) Exam #1 March 12, 2002 Name KEY SS# This exam is worth a total of 100 points. The number of points each question is worth is shown in parentheses after the question number. Good luck! 1. (5) Describe how the restriction-modification system works and what it is used for in bacterial cells. Restriction enzymes cut DNA at specific sequences. These sequences are also recognized by methylases, which methylate A or C residues. This methylation blocks restriction enzymes from cutting at their target sequences. Bacteria use the restriction-modification system to protect their own DNA and destroy invading phage DNA that is not methylated. 2. (3) Name three essential components of a plasmid vector. 1) Origin of replication 2) Site to insert foreign DNA (MCS) 3) Selectable marker (i.e. ampicillin resistance) 3. (2) Which of the following would be most appropriate for making a genomic library? a) pbr322 b) M13 phage c) cosmid answer - c 4. (6) α-amanatin is used at the concentrations below. For each concentration, state ALL of the RNA polymerases that will continue to function and what RNAs will be transcribed. Concentration Functional RNA polymerases RNAs transcribed 0.1µg/mL Pol I, Pol III rrna, 5SRNA/tRNA 200µg/mL Pol I rrna µg/mL Pol I, Pol II, Pol III rrna, mrna, 5SRNA/tRNA 1

2 5. (2) What happens to the C-terminal domain of RPB1, a core subunit of yeast RNA polymerase II, to stimulate transcription? It is phosphorylated. 6. (4) Pol II promoters can have up to four functional elements. Draw a generic Pol II promoter with all four elements and their positions relative to the start of transcription (+1) /-70-30/-25 upstream element TATA box Initiation downstream element region 7. (2) The typical RNA Pol I promoter contains a core element which overlaps the start of transcription and a upstream control element which lies between -156bp and -107bp upstream of the start of transcription. 8. (2) You have discovered a regulatory element within the first intron of a gene which increases the transcription rate when mutated so it is non-functional. What is the name for this type of element? Silencer/represseor/inhibitor 9. (5) Describe DNase footprint analysis and how it was used to show that TFIID binds to the TATA box. 1) Label TATA box containing promoter DNA such that only one strand is labeled 2) Incubate DNA with TFIID (experimental) or without TFIID (control) 3) Cut with dilute DNase so that you get ~1 cut/dna strand 4) Run DNA on gel to separate DNA fragments by size. 5) Postions where DNase couldn t cut due to protection by TFIID are seen as blank areas, which will be found at the position of the TATA box. 10. (2) What is the function of TAF II s at TATA-less promoters? TAF II s bind to the initiation region of to factors bound to other promoter elements so that TBP (TFIID) will be correctly positioned upstream of the start of transcription (where the TATA box is in TATA box containing genes). 2

3 11. (6) Match the following Pol II transcription factors with their function/activity. c TFIIA a) Phosphorylates Pol II d TFIIB b) Binds immediately before TFIIH b TFIIE c) Stabilizes TFIID binding e TFIIF d) Binds after TFIIA/TFIID a TFIIH e) Brings Pol II to preinitiation complexes 12. (4) Describe an experiment that shows TFIIH has helicase activity? A single strand plasmid (unlabelled) was hybridized to a labeled oligonucleotide and incubated with or without TFIIH. TFIIH unwinds the oligonucleotide with it s helicase activity. Thus when you run the TFIIH treated and untreated samples on a gel to separate by size, the sample without TFIIH will have a large labeled band representing plasmid and hybridized oligonucleotide, whereas the sample incubated with TFIIH will have a labeled band corresponding to the size of the oligonucleotide. 13. (3) Define what the parts of following acronym mean: TAF I 63. TAF TBP associated factor I Polymerase I kilodalton molecular weight 14. (4) Describe the steps involved in the initation of trna transcription including the relevant factors and promoter sequences. 1) TFIIIC binds to the internal promoter elements box a and box B 2) TFIIIC attracts TFIIIB to the upstream region where it binds 3) TFIIIB allows Pol III to bind upstream 4) Transcription begins and removes TFIIIC from the internal promoter 15. (3) TBP is associated with which transcription factors in Class I, Class II and Class III preinitiation complexes? Class I SLI Class II TFIID Class III TFIIIB 3

4 16. (3) A transcriptional activator binds to the sequence TAAGGTACCTTA as a homodimer. Which functional domains must this protein contain? 1) DNA binding domain, 2) dimerization domain, 3) activation domain 17. (4) The glucocorticoid receptor is a transcription factor that is typically found in the cytoplasm in the absence of glucocorticoid hormone. When glucocorticoid is present, describe the process by which glucocorticoid receptor activates transcription. 1) Glucocorticoid binds to the glucocorticoid receptor 2) Binding of glucocorticoid releases the hsp90 inhibitor from binding the receptor 3) The glucocorticoid receptor- glucocorticoid complex moves into the nucleus 4) The glucocorticoid receptor- glucocorticoid complex dimerizes and binds to enhancer elements to activate transcription 18. (2) Zinc fingers bind a zinc ion using two histidines amino acid residues in the alpha helix and two cysteines amino acid residues in the beta strand. 19. (4) bzip proteins contain a basic domain that uses positively charged amino acids to bind DNA, and a zipper domain consisting of alpha helices with leucines every seven amino acids which enable _interactions/dimerization_ between proteins. 20. (4) Derive the function of Gal4 in transcription activation based on the following run-off transcription experiment (i.e. What does Gal4 do and why?). Factors: D D,B B Run-off result Gal4: Incubation Wash Gal4 recruits TFIIB to the complex if Gal4 is bound first. Gal4 dependent Gal4: binding of TFIIB is also dependent Missing factors on TFIID. Incubation Run-off analysis NTPs 4

5 21. (2) A control region was deletion mapped and shown to have three enhancers: A, B and C. Enhancer binding proteins for site A and site C alone can stimulate transcription, but the protein that binds site B can not stimulate transcription. The protein that binds to site B is what type of transcription factor? The evidence is consistent with an architectural transcription factor. 22. (5) Describe the sequence of events that enable CREB to activate transcription. 1) CREB is present in the nucleus on the CRE enhancer element 2) PKA phosphorylates CREB 3) Phosphorylated CREB is bound by the mediator/co-activator CBP 4) CBP interacts with TAFII s to promote the formation of an initiation complex 5) Transcription is activated. 23. (2) The MAPK signal transduction pathway is initiated when growth factor binds to receptor. 24. (6) Match the term on the right that describes the term on the left. f Histone H3 a) formed by interactions among H1 histones e 50nm fiber b) histone octamer plus 146bp of DNA d Nucleosomes c) histone that is easily removed in low salt a Solenoids d) histone octamer, histone H1 and 200bp of DNA b Core particle e) comprised of 35kb 85kb loops c Histone H1 f) one of the core histones 25. (2) What is the function of an antirepressor? It removes nucleosomes from the promoter or prevents nucleosomes from binding. 5

6 26. (2) What assay would you use to reveal a nuclease free region in a promoter? DNAse hypersensitivity assay. 27. (5) Describe how an in vivo footprinting experiment is done and what it shows. In vivo footprinting is used to determine if nucleosomes are precisely positioned in a promoter of interest. Chromatin is treated with micrococcal nuclease at a concentration that cuts incompletely. Chromatin is then digested with restriction enzymes that bound a promoter fragment of interest. The chromatin fragments are size separated by gel electrophoresis and probed with a radioactive probe specific for you promoter region of interest. If bands are seen, that means that nucleosomes are precisely positioned within the promoter. If bands are not seen, then nucleosomes are randomly distributed along the promoter. Nucleosomes are typically precisely positioned in promoters of actively transcribed genes. 28. (4) Describe how histone acetylases function to activate transcription. They acetylate lysines on the N-terminal tails of H3 and H4, thus masking the + charges of the lysines. This masking of charge both decreases the strength of octamer binding to the DNA and decreases the interactions between nucleosomes. 29. (2) Sin3 and NcoR/SMRT are histone deacetylases that function to repress transcription. 6