MWF, 11:00-11:50 a.m., Chemistry 2201 Office: Chemistry 2505, 405-0058

Special Topics in Protein-Nucleic Acid Interaction

This course concerns advanced aspects of protein-nucleic acid interaction which are currently of interest in the literature. At the present time, I am intending to cover aspects of the four areas below. Depending on enrollment and the interests of the class, the syllabus is mutable; suggestions are welcome.

* The architecture of multi-protein DNA complexes involved in transcription and recombination: Methods, functional relevance, structures (Mu transposase, [[gamma]]d resolvase, enhanceosomes).

* Protein folding or reorganization upon interaction with nucleic acids; cooperative binding of proteins to DNA: Thermodynamics (hydrophobicity and heat capacity), functional relevance, examples (leucine zippers, BamH I, RNA-binding peptides, MAT a1/[[alpha]]2 heterodimer).

* DNA computing and nanotechnology: Building large, controlled structures out of DNA (DNA cubes and lattices); using the information-carrying capacity of DNA to perform massively parallel computations (the traveling salesman problem by PCR). Not bioinformatics.

* Spliceosome assembly/disassembly: The snRNP and pre-mRNA rearrangements required for pre-mRNA splicing. Role of ATPase activity in structural changes, regulation, and ensuring fidelity. Genetic and biochemical approaches. Functions of RNA and proteins.

The format of the class will be approximately 50% lectures, 40% discussion of required reading from the literature, and 10% student presentations on related topics. This oral presentation can be a critical evaluation of work in the literature or a research proposal, with discussion of methods, significance, and anticipated results. The presentation will be accompanied by a written document (< 10 pages), which will be due two weeks after the oral presentation. Required reading (approximately 20 papers) will be on reserve at the White Memorial Library. Some of the material will be review for those of you who have just taken BCHM 674, but there will be plenty of new stuff. In class discussions, I may assign individuals to provide background on particular aspects of the papers we discuss, and this will require additional literature work.

Office Hours: Tuesday, 2-3 p.m. and Wednesday, 2-3 p.m. in Chemistry 2505 (Biochemistry Wing)

Contacting me: jdkahn@umd.edu much preferred 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: Course materials will be available at http://www-chem.umd.edu/biochem/kahn/bchm669b

Grading will be based on class participation, on the quality of formal student presentations to the class, and on a short written paper relating to the oral presentation. Any second-year Biochemistry students would be well-advised to do their presentations relatively early in the semester. There will be no written examinations. The course grade will be based on performance relative to the other students and to my expectations (i.e., there's a curve but I'll move the cutoffs depending on how I feel about class performance).

Course Outline

Organization and general discussion of topics 1/29/95

I. Architecture

Introductory lecture:  Representative examples, why is it important                     1/31  
Methods:  Electrophoresis, microscopy, crystallography, ring-closure                     2/2  
Overview of site-specific recombination complexes:  Structure/function                   2/5  
Discussion of Rice and Steitz:  What goes into structural models?                        2/7  
Discussion of Baker and Mizuuchi:  What goes into biochemical models?                    2/9  
Introduction to eukaryotic transcription activation                                     2/12  
Overview of enhanceosomes:  TCR-[[alpha]], IFN-[[beta]]                                 2/14  
Discussion of Giese et al.:  Why are these complicated snups important?                 2/16  
Discussion of Thanos and Maniatis:  What do we really know?                             2/19  

Representative structural examples:  bZIP proteins, tat, Bam H I                        2/21  
Thermodynamics:  Spolar and Record model, relationship to protein folding               2/23  
Biological consequences of cooperativity, heterodimerization                            2/26  
Discussion of Weiss et al.:  Evidence for coordinated folding and binding               2/28  
Student presentations: 2 x 25 min                                                        3/1  
Discussion of Spolar and Record:  Theory and rationale                                   3/4  
Discussion of Li et al. adn Jin et al.:  Role of folding in sequence-specific            3/6  
recognition                                                                                   
Recognition of RNA by tat and rev peptides                                               3/8  
Synthesis of architecture and energetics: molecular matchmakers, chromatin remodeling   3/11  
Discussion of Sancar and Hearst:  How is the energy of ATP used to change outcomes      3/13  
Student presentations: 2 x 25 min                                                       3/15  
Discussion of Côte et al.:  How is chromatin manipulated in gene expression?            3/25  

Introduction:  Junctions, other shapes                                                  3/27  
Biochips and large-scale structures                                                     3/29  
Discussion of Robinson and Kallenbach:  What will it take?                               4/1  
Methods for designing DNA shapes                                                         4/3  
Discussion of Seeman (DNA cube):  How do we build things from DNA?                       4/5  
Student presentations: 2 x 25 min                                                        4/8  
Computing with DNA -- is it for real?                                                   4/10  
Discussion of Adelman and Lipton papers:  How does it really work, if it does?          4/12  
Discussion of McAdams and Shapiro:  Electrical analogies for biological circuits        4/15  
Student presentations: 2 x 25 min                                                       4/17  

Introduction to splicing                                                                4/19  
Biochemical and genetic methods                                                         4/22  
Discussion of Wassarman and Steitz                                                      4/24  
Discussion of Lesser and Guthrie                                                        4/26  
Student presentations: 2 x 25 min                                                       4/29  
Role of proteins in splicing                                                             5/1  
Discussion of Weeks and Cech                                                             5/3  
General function of NTP hydrolysis in fidelity                                           5/6  
Translational fidelity:  clocks, EF-Tu etc.                                              5/8  
Discussion of ...and Guthrie                                                            5/10  
Discussion of Yarus                                                                     5/13  

Reading List

This list may change as the semester progresses, but you will always have plenty of warning if it does. The required papers from the literature are listed on the following pages. 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 Company. 931 pp.

Lewin, B. (1994). Genes V. Oxford: Oxford University Press. 1272 pp.

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. 1163 pp.

Monographs for more in-depth discussion of particular topics:

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

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

Required Papers: If you use the original journals, be extremely careful not to damage them:

Adleman, L. M. (1994). "Molecular Computation of Solutions to Combinatorial Problems." Science 266, 1021-1024.

Baker, T. A., Mizuuchi, M., Savilahti, H., and Mizuuchi, K. (1993). "Division of Labor among Monomers within the Mu Transposase Tetramer." Cell 74, 723-733.

Chen, J. and Seeman, N. C. (1991). "Synthesis from DNA of a molecule with the connectivity of a cube." Nature 350, 631-633.

Du, W., Thanos, D., and Maniatis, T. (1993). "Mechanisms of Transcriptional Synergism between Distinct Virus-Inducible Enhancer Elements." Cell 74, 887-898.

Echols, H. (1986). "Multiple DNA-Protein Interactions Governing High-Precision DNA Transactions." Science 233, 1050-1056.

Giese, K. and Grosschedl, R. (1993). "LEF-1 contains an activation domain that stimulates transcription only in a specific context of factor-binding sites." EMBO J. 12(12), 4667-4676.

Giese, K., Kingsley, C., Kirshner, J. R., and Grosschedl, R. (1995). "Assembly and function of a TCR[[alpha]] enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions." Genes Dev. 9, 995-1008.

Grosschedl, R. (1995). "Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination." Curr. Opinion Cell Biol. 7, 362-370.

Grosschedl, R., Giese, K., and Pagel, J. (1994). "HMG domain proteins: architectural elements in the assembly of nucleoprotein structures." Trends Genetics 10(3), 94-100.

Jin, Y., Mead, J., Li, T., Wolberger, C., and Vershon, A. K. (1995). "Altered DNA Recognition and Bending by Insertions in the [[alpha]]2 Tail of the Yeast a1/[[alpha]]2 Homeodomain Heterodimer." Science 270, 290-293.

Li, T., Stark, M. R., Johnson, A. D., and Wolberger, C. (1995). "Crystal Structure of the MATa1/MAT[[alpha]]2 Homeodomain Heterodimer Bound to DNA." Science 270, 262-269.

Lipton, R. J. (1995). "DNA Solution of Hard Computational Problems." Science 268, 542-545.

McAdams, H. H. and Shapiro, L. (1995). "Circuit Simulation of Genetic Networks." Science 269, 650-656.

Rice, P. A. and Steitz, T. A. (1994). "Model for a DNA-mediated synaptic complex suggested by crystal packing of gamma delta resolvase subunits." EMBO J. 13, 1514-1524.

Robinson, B. H. and Seeman, N. C. (1987). "The design of a biochip: a self-assembling molecular-scale memory." Protein Engineering 1(4), 295-300.

Spolar, R. S. and Record, M. T., Jr. (1994). "Coupling of local folding to site-specific binding of proteins to DNA." Science 263(5148), 777-84.

Weiss, M. A. (1990). "Thermal Unfolding Studies of a Leucine Zipper Domain and Its Specific DNA Complex: Implications for Scissor's Grip Recognition." Biochemistry 29, 8020-8024.

Weiss, M. A., Ellenberger, T., Wobbe, C. R., Lee, J. P., Harrison, S. C., and Struhl, K. (1990). "Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA." Nature 347, 575-578.

Zhang, Y. and Seeman, N. C. (1994). "Construction of a DNA-Truncated Octahedron." J. Am. Chem. Soc. 116, 1661-1669.