Biochemistry 674.
Fall, 1999: Nucleic Acids
Asst. Prof. Jason D. Kahn
Office: Chemistry 2505, 405-0058
TuTh, 8:00-9:15 a.m., Chemistry 2201

This course concerns the structure and function of nucleic acids and the mechanisms of nucleic acid transactions, with an emphasis on molecules rather than molecular genetics (see MOCB 630). Topics will include those in the list below, and I am willing to adjust the material according to the interests of the class.

  • Chemistry and structure of DNA and RNA, from nucleotides to chromosomes, and some methods for studying, synthesizing, sequencing and manipulating nucleic acids. Bioinformatics.
  • Interactions between nucleic acids and ligands such as cations, drugs, and especially proteins
  • Selected aspects of the biochemistry and regulation of DNA replication, transcription, recombination, and repair, and how these processes interact with each other
  • RNA splicing, RNA catalysis, translation, and selection-amplification methods.
    •      Required papers from the literature will be assigned for each lecture (may be read after the lecture), as indicated on the Course Outline below. Most of the papers will be included in a course packet, cost $25., and all papers will be on reserve at the White Memorial Chemistry Library. In addition, two textbooks are strongly recommended for the course, for background and for reference in your research careers. I will provide handouts with some figures, hints on what you should take away from the assigned reading, and additional entries into the literature. Some of these additional sources, especially books, are on reserve.

    There will be two 75-minute exams (100 pts each) and a two hour final (200 pts). Exams will emphasize lecture material, with some coverage of key concepts from the reading. You will be asked to design and interpret experiments as well as to recapitulate assigned material. Review sessions will be held, past exams will be on reserve, and hints on what you should get out of the reading will be provided on the course web site and the handouts. I encourage questions and discussion in class, but class participation does not affect grading. If you absolutely must miss an exam, you must call me in advance or within 24 hours after the exam, and you must also present a valid University excuse, in order to be eligible for the assignment of a grade based on the remaining two exams. If you miss the final or both hour exams, you will receive a failing grade. The exams are quite difficult, but in the past I have had few complaints about final grades. Your course grade will be based on exam performance relative to a curve and to my expectations.

  • Office hours: Wednesday, 2-3 p.m. and Thursday, 1-2 p.m. in Chemistry 2505 (Biochemistry Wing). There is no TA for the course
  • Contacting me: jdkahn@umd.edu is preferable to 405-0058. Please do not drop in to my office or lab, but I will be happy to set up appointments outside of office hours if necessary
  • Web site etc.: Course materials are available at http://www-chem.umd.edu/biochem/kahn/bchm674. This is primarily meant as a source for molecular graphics tutorials. There will also be an e-mail reflector for the class.
  • Recommended texts, available at the University Book Center:

    • Calladine, C. R. & Drew, H. R. (1997). Understanding DNA: The Molecule and How It Works. 2nd ed., Academic Press, San Diego. Opinionated, but with interesting physical insights.
    Weaver, R. F. (1999). Molecular Biology. 1st ed., WCB/McGraw-Hill, Boston. Excellent source for historical and modern experiments.

    Course Outline

    I. Nucleic Acid Sequence, Structure, and Chemistry (10 lectures)

    1. Nucleic acid building blocks 9/2/99

    Nucleotide structure, primary structure, chemical stability, nomenclature

    Required reading: None

    2. Structures of double helices 9/7

    A, B, and Z form helices, base pairing and hydrogen bonding

    Watson and Crick, 1953; Dickerson, 1983

    3. DNA and RNA hybridization and thermodynamics 9/9

    Base-pair stability rules, melting, hybridization, hypochromism

    Freier et al., 1986 [X, = not in course photocopy book]

    4. RNA structure and triple helices 9/14

    Tertiary structure and tRNA, prediction of RNA folding, antisense

    Moser and Dervan, 1987

    5. Sequencing and synthesis of DNA and RNA 9/16

    Maxam-Gilbert and Sanger sequencing, chemical and enzymatic synthesis, bioinformatics

    Fleischmann et al., 1995

    6. DNA bending, flexibility, and cyclization 9/21

    Bending and twisting flexibility, sequence-directed bending, methods for detection and quantitation

    Zinkel and Crothers, 1987; Kahn and Crothers, 1992

    7. Drug and cation binding, chemical probing methods 9/23

    Intercalation, groove-binding, ion atmosphere, reactivity of nucleotides, altered backbone chemistries

    Siebenlist et al., 1980

    8. Topology, supercoiling, topoisomerases 9/28

    Linking number, superhelix structure, topo reaction mechanisms, knots and catenanes

    Bauer et al., 1981

    9. Enzymatic manipulation of nucleic acids 9/30

    Restriction enzymes, nucleases, radiolabeling, basic genetic engineering, polymerases, PCR

    Arnheim and Levenson, 1990

    10. Catch-up day 10/4

    II. General Features of Protein-Nucleic Acid Interaction (4 lectures)

    11. Biochemical methods for studying complexes 10/7

    Binding curves, gel mobility shift, footprinting/interference, crosslinking, filter binding

    Fried and Crothers, 1981 [X]

    12. Protein structural motifs for nucleic acid binding 10/12

    Helix-turn-helix, zinc fingers, bZIP proteins, TBP, hnRNP, etc.

    Harrison, 1991; Nikolov et al., 1995

     -- EXAM I -- Covers section I and lecture 11. 10/14 13. Sequence- and structure-specific recognition of nucleic acids 10/19

    Major groove vs. minor groove, hydrogen bonding, direct vs. indirect readout, deformability, RNA recognition

    Seeman et al., 1976

    14. Chromosome structure 10/21

    Nucleosomes, chromatin, higher-order structure, telomeres

    Schild et al., 1993 [X]

    III. DNA Transactions (8 lectures)

    15. DNA replication: fundamental mechanisms 10/26

    Polymerization reaction mechanisms, fidelity, structure

    Brutlag and Kornberg, 1972

    16. In vitro genome replication 10/28

    Origin recognition and polymerase holoenzymes in E. coli and SV40; the cell cycle.

    Waga and Stillman, 1994, Naktinis et al., 1996

    17. Transcription: fundamental mechanisms 11/2

    RNA polymerases, transcription cycle, transcription bubble, supercoiling

    Nudler et al., 1997; Liu and Wang, 1987

    18. Regulation in prokaryotes; looping, tracking 11/4

    Paradigms: lac operon, araC, ntrC. Searching mechanisms.

    Echols, 1990; Lobell and Schleif, 1990

    19. Transcription in eukaryotes 11/9

    Holoenzyme vs. initiation complex assembly, activators, enhancers, chromatin, recruitment

    Struhl, 1999 [X]

    20. Recombination 11/11

    Holliday junctions, l phage integration and excision, recABCD

    Nash, 1990

     -- EXAM II -- Covers through Lecture 19. 11/16 21. DNA repair 11/18

    BER, NER, mismatch repair, cancer

    Sancar, 1995

    22. "Interprocess Communication" 11/23

    Review of regulatory and biochemical connections among replication, transcription, repair

     -- Thanksgiving-- No lecture 11/25

    IV. RNA Transactions (5 lectures)

    23. Catalytic RNA 11/30

    Self-splicing RNA, ribozymes, origin of life

    Zaug and Cech, 1986; Uhlenbeck, 1987

    24. RNA splicing and degradation 12/2

    Splicing mechanisms, control of mRNA lifetime

    Nilsen, 1994

    25. Translation 12/7

    Chemistry of protein biosynthesis, ribosome structure

    26. Selection-amplification methods for nucleic acids 12/9

    Selection of optimal DNA and RNA ligands or catalysts, in vitro evolution

    Ellington and Szostak, 1990

    27. Review and/or catch up day. 12/14

     -- FINAL EXAM-- Comprehensive Saturday, 12/18/99, 8:00-10:00 a.m., Chem. 2201

    Reading List

    This list may change as the semester progresses. In the required papers, I have tried to strike a balance among review articles, classic papers, and current research. Please let me know if there are difficulties with the amount or depth of the reading.

    General texts for further reading and background:

    Adams, R. L. P., Knowler, J. T., and Leader, D. P. (1992). The Biochemistry of the Nucleic Acids. 11th ed. London: Chapman & Hall. Fact compendium.

    Kornberg, A. and Baker, T. A. (1992). DNA Replication. 2nd ed. New York: W.H. Freeman and Co. Focuses on classical biochemistry experiments.

    Lewin, B. (1997). Genes VI. Oxford: Oxford University Press. Deeply flawed but relatively up-to-date.

    Schleif, R. (1993). Genetics and Molecular Biology. 2nd ed. Baltimore: The Johns Hopkins University Press. Eclectic, emphasizing experiments leading to conclusions.

    Watson, J. D., Hopkins, N. H., Roberts, J. W., Steitz, J. A., and Weiner, A. M. (1987). Molecular Biology of the Gene. 4th ed. Menlo Park, CA: The Benjamin/Cummings Publishing Company, Inc.

    Monographs for more in-depth discussion of particular topics:

    Bates, A. D. and Maxwell, A. (1993). DNA Topology. Oxford: IRL Press at Oxford University Press. 114 pp. Excellent short monograph on this difficult topic.

    Ptashne, M. (1992). A Genetic Switch: Phage l and Higher Organisms. 2nd ed. Cambridge, MA: Cell Press and Blackwell Scientific. 192 pp. Heuristics of gene regulation.

    Saenger, W. (1984). Principles of Nucleic Acid Structure. New York: SpringeróVerlag. 556 pp. Very technical and detailed.

    Steitz, T. A. (1993). Structural Studies of Protein-Nucleic Acid Interaction: The sources of sequence-specific binding. Cambridge, England: Cambridge University Press. 79 pp. Good pictures.

    Travers, A. (1993). DNA-Protein Interactions. London: Chapman & Hall. 180 pp. And DNA structure.

    Wolffe, A. (1999). Chromatin: Structure and Function. 3rd ed. San Diego: Academic Press, Inc. 400 pp. Covers from structure to biology.

    Required Papers:

    The papers indicated by [X] are not in the class book due to copyright or timing issues.

    Arnheim, N. and Levenson, C. H. (1990). "Polymerase Chain Reaction." Chem. & Eng. News (October 1), 38-47.

    Bauer, W. R., Crick, F. H. C., and White, J. H. (1980). "Supercoiled DNA." Scientific American 243(1), 118-133.

    Brutlag, D. and Kornberg, A. (1972). "Enzymatic Synthesis of Deoxyribonucleic Acid: XXXVI. A proofreading function for the 3¢Æ 5¢ exonuclease activity in deoxyribonucleic acid polymerases." J. Biol. Chem. 247, 241-248.

    Chen, J.-L., Attardi, L. D., Verrijzer, C. P., Yokomori, K., and Tjian, R. (1994). "Assembly of Recombinant TFIID Reveals Differential Coactivator Requirements for Distinct Transcriptional Activators." Cell79, 93-105.

    Dickerson, R. E. (1983). "The DNA Helix and How It Is Read." Sci. Am. (December), 94-111.

    Echols, H. (1990). "Nucleoprotein Structures Initiating DNA Replication, Transcription, and Site-specific Recombination." J. Biol. Chem. 265, 14697-14700.

    Ellington, A. D. and Szostak, J. W. (1990). "In vitro selection of RNA molecules that bind specific ligands." Nature 346, 818-822.

    Fleischmann, R. D., Adams, M. D., White, O., many others & Venter, J. C. (1995). "Whole-Genome Random Sequencing and Assembly of Haemophilus influenzae Rd." Science 269, 496-512.

    [X] Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986). "Improved free-energy parameters for predictions of RNA duplex stability." Proc. Natl. Acad. Sci. USA 83, 9373-9377.

    [X] Fried, M. and Crothers, D. M. (1981). "Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis." Nucleic Acids Res. 9, 6505-6525.

    Harrison, S. C. (1991). "A structural taxonomy of DNA-binding domains." Nature 353, 715-719.

    Kahn, J. D. and Crothers, D. M. (1992). "Protein-induced bending and DNA cyclization." Proc. Natl. Acad. Sci. USA 89, 6343-7.

    Liu, L. F. and Wang, J. C. (1987). "Supercoiling of the DNA Template During Transcription." Proc. Nat. Acad. Sci. USA84, 7024-7027.

    Lobell, R. B. and Schleif, R. F. (1990). "DNA Looping and Unlooping by AraC Protein." Science 250, 528-32.

    Moser, H. E. and Dervan, P. B. (1987). "Sequence-Specific Cleavage of Double Helical DNA by Triple Helix Formation." Science238, 645-650.

    Naktinis, V., Turner, J. & OíDonnell, M. (1996). "A Molecular Switch in a Replication Machine Defined by an Internal Competition for Protein Rings." Cell 84, 137-145.

    Nash, H. A. (1990). "Bending and supercoiling of DNA at the attachment site of bacteriophage l." Trends Biochem. Sci. 15, 222-227.

    Nikolov, D. B., Chen, H., Halay, E. D., Usheva, A. A., Hisatake, K., Lee, D. K., Roeder, R. G. & Burley, S. K. (1995). "Crystal structure of a TFIIB-TBP-TATA element ternary complex." Nature377, 119-128.

    Nilsen, T. W. (1994). "RNA-RNA Interactions in the Spliceosome: Unraveling the Ties That Bind." Cell 78, 1-4.

    Nudler, E., Mustaev, A., Lukhtanov, E., and Goldfarb, A. (1997). "The RNA-DNA Hybrid Maintains the Register of Transcription by Preventing Backtracking of RNA Polymerase." Cell 89, 33-41.

    Sancar, A. (1995). "Excision Repair in Mammalian Cells." J. Biol. Chem. 270, 15915-15918.

    [X] Schild, C., Claret, F.-X., Wahli, W., and Wolffe, A. P. (1993). "A nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in vitro." EMBO J. 12, 423-433.

    Seeman, N. C., Rosenberg, J. M., and Rich, A. (1976). "Sequence-specific recognition of double helical nucleic acids by proteins." Proc. Natl. Acad. Sci. USA 73, 804-808.

    Siebenlist, U., Simpson, R. B., and Gilbert, W. (1980). "E. coli RNA Polymerase Interacts Homologously with Two Different Promoters." Cell 20, 269-281.

    [X] Struhl, K. (1999). "Fundamentally Different Logic of Gene Regulation in Eukaryotes and Prokaryotes." Cell 98, 1-4.

    Uhlenbeck, O. C. (1987). "A Small Catalytic Oligoribonucleotide." Nature 328, 596-600.

    Waga, S. and Stillman, B. (1994). "Anatomy of a DNA Replication fork revealed by reconstitution of SV40 DNA replication in vitro." Nature 369, 207-212.

    Watson, J. D. and Crick, F. H. C. (1953). "Molecular Structure of Nucleic Acids: A structure for deoxyribose nucleic acid." Nature 171, 737-738.

    Zaug, A. J. and Cech, T. R. (1986). "The Intervening Sequence RNA of Tetrahymena Is an Enzyme." Science 231, 470-475.

    Zinkel, S. S. and Crothers, D. M. (1987). "DNA bend direction by phase sensitive detection." Nature 328, 178-181.