Jump to Course Outline (lecture schedule)
Biochemistry 674. Fall, 2000: Nucleic Acids Assoc. Prof. Jason D. Kahn
TuTh, 8:00-9:15 a.m., Chemistry 2201 Office: Chemistry 2505, 405-0058
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:
• 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 some lectures (may be read after the lecture), as indicated on the Course Outline below. 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 also provide occasional 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), a short (7-10 pp.) paper (100 pts), and a two hour final examination (150 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 and past exams will be on reserve. The paper will be an examination of the historical development of our understanding of a particular topic, with emphasis on critical experiments and how they caused reinterpretation of earlier work. 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 and the paper. 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 and paper performance relative to a curve and to my expectations. I expect strict adherence to the University’s Code of Academic Integrity.
Office hours: Wednesday, 1:30-2:30 p.m. and Thursday, 2-3 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:
Bloomfield, V.A., Tinoco, I., Jr. and Crothers, D.M. (2000). Nucleic Acids: Structure, Properties and Functions. University Science Books, Sausalito CA. Biophysical chemistry.
Weaver, R. F. (1999). Molecular Biology. 1st ed., WCB/McGraw-Hill, Boston. Excellent source for historical and modern experiments.
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I. |
Nucleic Acid Sequence, Structure, and Chemistry (10 lectures) |
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1. |
Nucleic acid building blocks |
8/31/00 |
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Nucleotide structure, primary structure, chemical stability, nomenclature |
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2. |
Structures of double helices |
9/5/00 |
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A, B, and Z form helices, base pairing and hydrogen bonding |
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Watson and Crick, 1953; Dickerson, 1983 |
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3. |
DNA and RNA hybridization and thermodynamics |
9/7/00 |
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Base-pair stability rules, melting, hybridization, hypochromism, gene chips |
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Holstege et al., 1999 |
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4. |
RNA structure and triple helices |
9/12/00 |
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Tertiary structure and tRNA, prediction of RNA folding, antisense |
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Moser and Dervan, 1987 |
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5. |
Sequencing and synthesis of DNA and RNA |
9/14/00 |
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Maxam-Gilbert and Sanger sequencing, chemical and enzymatic synthesis, bioinformatics |
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Fleischmann et al., 1995 |
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6. |
DNA bending, flexibility, and cyclization |
9/19/00 |
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Bending and twisting flexibility, sequence-directed bending, methods for detection and quantitation |
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Zinkel and Crothers, 1987; Kahn and Crothers, 1992 |
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7. |
Drug and cation binding, chemical probing methods |
9/21/00 |
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Intercalation, groove-binding, ion atmosphere, reactivity of nucleotides, altered backbone chemistries |
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Siebenlist et al., 1980 |
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8. |
Topology, supercoiling, topoisomerases |
9/26/00 |
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Linking number, superhelix structure, topo reaction mechanisms, knots and catenanes |
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Bauer et al., 1980 |
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9. |
Enzymatic manipulation of nucleic acids |
9/28/00 |
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Restriction enzymes, nucleases, radiolabeling, basic genetic engineering, polymerases, PCR |
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10. |
Catch-up day |
10/3/00 |
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II. |
General Features of Protein-Nucleic Acid Interaction (4 lectures) |
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11. |
Biochemical methods for studying complexes |
10/5/00 |
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Binding curves, gel mobility shift, footprinting/interference, crosslinking, filter binding |
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12. |
Protein structural motifs for nucleic acid binding |
10/10/00 |
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Helix-turn-helix, zinc fingers, bZIP proteins, TBP, hnRNP, etc. |
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Harrison, 1991; Nikolov et al., 1995 |
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| EXAM I: Covers through Section I. | 10/12/00 | |
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13. |
Sequence- and structure-specific recognition of nucleic acids |
10/17/00 |
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Major groove vs. minor groove, hydrogen bonding, direct vs. indirect readout, deformability, RNA recognition |
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Seeman et al., 1976 |
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14. |
Chromosome structure |
10/19/00 |
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Nucleosomes, chromatin, higher-order structure, telomeres |
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Luger et al., 1997 |
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III. |
DNA Transactions (8 lectures) |
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15. |
DNA replication: fundamental mechanisms |
10/24/00 |
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Polymerization reaction mechanisms, fidelity, structure |
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Brutlag and Kornberg, 1972 |
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16. |
In vitro genome replication |
10/26/00 |
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Origin recognition and polymerase holoenzymes in E. coli; the cell cycle. |
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Naktinis et al., 1996 |
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17. |
Transcription: fundamental mechanisms |
10/31/00 |
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RNA polymerases, transcription cycle, transcription bubble, supercoiling |
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Nudler et al., 1997; Liu and Wang, 1987 |
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18. |
Regulation in prokaryotes; repression, activation, looping |
11/2/00 |
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Paradigms: lac operon, araC, ntrC. Searching mechanisms. |
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Echols, 1990; Lobell and Schleif, 1990 |
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19. |
Transcription in eukaryotes |
11/7/00 |
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Holoenzyme vs. initiation complex assembly, activators, enhancers, chromatin, recruitment |
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Struhl, 1999; Cosma et al., 1999 |
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20. |
Recombination |
11/9/00 |
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Holliday junctions, l phage integration and excision, recABCD |
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Nash, 1990 |
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| Paper Topics Due | 11/9/00 | |
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21. |
DNA repair |
11/14/00 |
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BER, NER, mismatch repair, cancer |
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Sancar, 1995 |
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| EXAM II: Covers through Lecture 20. | 11/16/00 | |
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22. |
"Interprocess Communication" |
11/21/00 |
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Review of regulatory and biochemical connections among replication, transcription, repair |
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| Thanksgiving: No lecture | 11/23/00 | |
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IV. |
RNA Transactions (5 lectures. | |
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23. |
Catalytic RNA |
11/28/00 |
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Self-splicing RNA, ribozymes, origin of life |
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Uhlenbeck, 1987 |
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24. |
RNA splicing and degradation |
11/30/00 |
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Splicing mechanisms, control of mRNA lifetime |
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Nilsen, 1994 |
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| Paper Due | 12/5/00 | |
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25. |
Translation |
12/5/00 |
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Chemistry of protein biosynthesis, ribosome structure |
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Nissen et al., 2000 |
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26. |
Selection-amplification methods for nucleic acids |
12/7/00 |
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Selection of optimal DNA and RNA ligands or catalysts, in vitro evolution |
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Ellington and Szostak, 1990 |
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27. |
Review and/or catch up day. |
12/12/00 |
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FINAL EXAM: Emphasizes 21-27:
Sat., 12/16/00, 8:00-10:00 a.m., Chem. 2201
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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:
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. (1999). Genes VII. 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.
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: Springer—Verlag. 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:
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.
Cosma, M. P., Tanaka, T. and Nasmyth, K. (1999). "Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter." Cell 97(3), 299-311.
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 and Venter, J. C. (1995). "Whole-Genome Random Sequencing and Assembly of Haemophilus influenzae Rd." Science 269, 496-512.
Harrison, S. C. (1991). "A structural taxonomy of DNA-binding domains." Nature 353, 715-719.
Holstege, F. C. P., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S. and Young, R. A. (1999). "Dissecting the Regulatory Circuitry of a Eukaryotic Genome." Cell 95, 717-728.
Kahn, J. D. and Crothers, D. M. (1992). "Protein-induced bending and DNA cyclization." Proc. Natl. Acad. Sci. USA 89, 6343-6347.
Liu, L. F. and Wang, J. C. (1987). "Supercoiling of the DNA Template During Transcription." Proc. Nat. Acad. Sci. USA 84, 7024-7027.
Lobell, R. B. and Schleif, R. F. (1990). "DNA Looping and Unlooping by AraC Protein." Science 250, 528-532.
Luger, K., Mäder, A. W., Richmond, R. K., Sargent, D. F. and Richmond, T. J. (1997). "Crystal structure of the nucleosome core particle at 2.8 Å resolution." Nature 389, 251-260.
Moser, H. E. and Dervan, P. B. (1987). "Sequence-Specific Cleavage of Double Helical DNA by Triple Helix Formation." Science 238, 645-650.
Naktinis, V., Turner, J. and O’Donnell, 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. and Burley, S. K. (1995). "Crystal structure of a TFIIB-TBP-TATA element ternary complex." Nature 377, 119-128.
Nilsen, T. W. (1994). "RNA-RNA Interactions in the Spliceosome: Unraveling the Ties That Bind." Cell 78, 1-4.
Nissen, P., Hansen, J., Ban, N., Moore, P. B. and Steitz, T. A. (2000). "The Structural Basis of Ribosome Activity in Peptide Bond Synthesis." Science 289, 920-930.
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.
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.
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.
Watson, J. D. and Crick, F. H. C. (1953). "Molecular Structure of Nucleic Acids: A structure for deoxyribose nucleic acid." Nature 171, 737-738.
Zinkel, S. S. and Crothers, D. M. (1987). "DNA bend direction by phase sensitive detection." Nature 328, 178-181.