Molecular Biology (BIOL 4320) Exam #2 April 22, 2002

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1 Molecular Biology (BIOL 4320) Exam #2 April 22, 2002 Name 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. (3) Draw a diagram of an intron showing the conserved splicing sequences and where they reside. 5 splice site Branch point 3 splice site Exon 1 GU A AG Exon nt 2. (4) Diagram or describe the two step mechanism of splicing and the intermediates formed after each step. OH Exon1 pgu A AGp Exon2 1 G p Exon 1 OH + A AGp Exon 2 2 Exon 1 p Exon 2 + A AG-OH 3. (5) Match the U RNA with its function in the splicing mechanism. e U6 a) Binds to and sequesters U6 until splicing has commenced d U5 b U1 c U2 a U4 b) The first U RNA to bind the 5 splice site c) Base pairs with sequences around the branch point d) Base pairs with the last base of exon1 and the first base of exon2 e) The second U RNA to bind the 5 splice site 1

2 4. (2) How would you show that an intron is committed to be spliced? You could preincubate labeled pre-mrna with an SR protein such as SC35 and compete with an excess of unlabelled cometitor pre-mrna. If the competitor does not compete with the pre-incubated labeled pre-mrna, then splicing should occur, showing the labeled pre-mrna was committed to be spliced. 5. (2) Some introns are self-splicing, showing that RNA can act as a catalyst/enzyme. 6. (2) The 5 end of mrnas are capped by 7-methyl Guanosine that is linked to the mrna through a 5 5 linkage. 7. (4) List four functions of the 5 mrna cap. 1) It stabilizes/protects the mrna 2) It enhances the translation of the mrna 3) It is required for movement of the mrna from nucleus to cytoplasm 4) It is required for removing the first intron 8. (3) Draw a diagram showing 1) the sequences required for poly-adenylation of mrna, 2) where they reside in the mrna and 3) which factors bind to these sequences. 5 AAUAAA (~20nt) GUGUGUUUUUUUUU 3 Cleavage site SPSF binds to the AAUAAA sequence and CstF binds to the GU/U rich region 9. (2) PolyAdenylation enhances translation by enabling more efficient recruitment of mrna into polysomes. 10. (4) Describe how transferrin receptor (TfR) mrna is stabilized when iron levels drop inside the cell. Be sure to include all relevant factors and sequences. When iron levels are high, TfR mrna levels are low because IRE elements in the 3 UTR are susceptible to cleavage by RNAses, thus destabilizing the mrna. As iron levels drop, IRE binding protein loses it s bound iron, which allows the IRE binding protein to bind to the IRE elements in the TfR 3 UTR. Binding of IREs by IRE binding protein protects the IREs from cleavage by RNAses, thus stabilizing TfR mrna and allowing it to accumulate to high levels. 2

3 11. (4) Identify the best translation start site in the mrna below. 5 AUGGCUUAUGCUCGTCCGCCAUGGCUUUAAGUACGGUAAAUGCUACA 3 This AUG is the only one in a good Kozak consensus 12. (4) In the absence of heme, heme controlled repressor phosphorylates eif2 so that it binds very tightly to eif2b. Why does this block translation? When phosphorylated eif2 binds tightly to eif2b, it stays bound to eif2b and does not allow eif2b to exchange GTP for GDP for other eif2s, thus rendering eif2s inactive. 13. (8) Match the following translation initiation factors with their function/activity. d eif1/1a c eif4e b eif3 e eif2 f eif4a h eif6 a eif5 g eif4g a) Promotes binding of 60S subunit to the 48S complex b) Binds 40S subunit to block premature 60S subunit binding c) Binds to the 5 cap of the mrna d) Allows ribosome to scan to the Kozak AUG e) Binds initiating aminoacyl trna to the 40S subunit f) An RNA helicase g) Adaptor that recruits 40S subunits to the mrna h) Binds the 60S subunit to block premature 48S complex binding 3

4 14. (4) Describe how translation of ferretin is stimulated when iron levels are high. Be sure to include all relevant factors and sequences. Ferretin mrna has an IRE in the 5 UTR. When iron levels are low, the IRE is bound by IRE binding protein, thus blocking translation and lowering the levels of ferretin. When iron levels increase, iron binds to IRE binding protein, thus releasing IRE binding protein from the IRE and relieving the block on translation. 15. (2) EF-Tu/Ts are required for binding aminoacyl-trnas to the ribosome. 16. (4) Describe two proofreading mechanisms that occur during translation elongation? The first mechanism occurs at the binding step of the ternary complex to the A site, where binding of the ternary complex dissociates if the aminoacyl-trna is not correct. The second step occurs after binding of the ternary complex to the A site, where aminoacyl-trna is released before peptide bond formation occurs if it the incorrect aminoacyl trna. 17. (3) Peptidyl transferase activity is catalyzed by a conserved A residue at position 2486 in the 23S rrna. 18. (4) During translation, EF-G promotes translocation of the mrna by 3 (number) nucleotides in a process that requires hydrolysis of GTP. 19. (4) RF1 and RF2 mimic trnas by recognizing the stop codons UAG, UGA, or UAA in the A site. RF3 is a ribosome-dependent GEF that helps RF1 and RF2 bind to the stop codons. 4

5 20. (4) Label the following structural features of 30S and 50Ss ribosomal subunits. Central protuberance Ridge Stalk platform 21. (5) Label the cloverleaf trna structure below. Acceptor stem D loop T loop Variable loop Anticodon loop 22. (4) The anticodon and the acceptor stem are the two primary sites on trnas that are recognized by aminoacyl-trna synthetases. 5

6 23. (5) Describe how aminoacyl-trna synthetases select the correct amino acid. Make sure and include any intermediates that are formed. Initially there is a size screen that does not allow amino acids larger than the correct amino acid for the specific amnioacyl trna synthetase into the activation site. The correct amino acid and smaller amino acids enter the activation site and are activated via adenylation. The adenylated amino acids that are smaller than the correct amino acid then enter the editing site, where they are deadenylated and removed from the aminoacyl trna synthetase. Since the correct amino acid can t enter the editing site, it is added to the trna, thus forming a charged aminoacyl trna. 24. (4) Name four advantages for using BAC vectors over YAC vectors for making human genomic DNA libraries. 1) Inserts can readily be cloned into BAC vectors 2) BAC DNA is easy to isolate in large quantities 3) They are stable transformants in E. coli 4) They have comparatively very low rates of insert scrambling. 25. (4) When calculating the number of genes in a sequenced genome, the total number of annotated genes is comprised of what four categories of genes? 1) Known genes, 2) related genes, 3) predicted genes and 4) pseudogenes. 26. (2) The human genome is estimated to contain ~30,000 genes, but because of alternative splicing, these genes are thought to encode ~100,000 proteins. 27. (2) Name two techniques you could use to identify exons within a genomic region. 1) Exon trapping 2) Identifying unmethylated CpG islands using HpaII. 28. (2) Microarrays are used to determine the state of gene expression. 6