Schedule for 501 Gene Expression Section # Lecture Date Lecturer Lecture Title

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1 Schedule for 501 Gene Expression Section 2008 Section Director, Dr. Peter Zassenhaus, Meets in LRC105 from 9-10 AM or from 9-11 AM on dual lecture days # Lecture Date Lecturer Lecture Title 1 Tuesday, October 7 Zassenhaus Overview and Bacterial Gene Expression 2 Wednesday, October 8 Zassenhaus Bacterial Gene Expression 3 Thursday, October 9 Zassenhaus Bacterial Gene Expression 4 Friday, October 10 (9 AM) Zassenhaus Eukaryotic Gene Regulation 5 Friday, October 10(10 AM) Zassenhaus Eukaryotic Gene Regulation 6 Monday, October 13 Dorsett RNA Processing 7 Tuesday, October 14 Eissenberg Chromatin Structure and Transcription I 8 Wednesday, October 15 Eissenberg Chromatin Structure and Transcription II 9 Thursday, October 16 Eissenberg Transcriptional Repression 10 Friday, October 17 (9 AM) Zassenhaus Summary and Review of Eukaryotic Gene Regulation 11 Friday, October 17 (10 AM) Eliceiri Micro RNAs and RNAi 12 Monday, October 20 Chang Protein Synthesis I 13 Tuesday, October 21 Chang Protein Synthesis II 14 Wednesday, October 22 Skowyra Protein Folding and Quality Control 15 Thursday, October 23 Skowyra Targeted Proteolysis as a Main Regulatory Mechanism of Gene Expression I 16 Friday, October 24 Note: meet in Schwitalla 409 Skowyra Targeted Proteolysis as a Main Regulatory Mechanism of Gene Expression II 17 Monday, October NOON EXAM 1

2 Transcriptional Regulation Section on Gene Expression Tuesday, October 7 Friday, October 10 Zassenhaus, Doisy Research Center, Room 663 or 603, Ext , Zassenp@slu.edu Part I: Bacterial Transcriptional Regulation Readings: On reserve in the Library are copies of the book Genes and Signals by Mark Ptashne and Alexander Gann. Before class starts, please read Chapter 1, pages This is a very readable, small page size book with lots of good figures, not heavy on facts, but superbly rich on ideas and explanations. This one chapter explains ALL of transcription, gene regulation, AND cell signaling in all life forms! Once you understand the principles presented here, then all the rest is just filling in the details. Our goal in class will be to learn those principles. Before Class on Thursday, finish reading Chapter 1, pages During Part 1, we will focus on how the binding properties of transcriptional regulators determine how regulation works. Therefore, it will be helpful if you review protein-protein interactions from earlier in the course, particularly the quantitative aspects of measuring protein binding i.e., the math of binding equilibria. Over the course of the first three lectures (Tuesday thru Thursday) we will cover: 1. Structure and function of the bacterial RNA polymerase 2. Regulated Recruitment of transcriptional regulators: Protein-DNA Interactions Detecting physiological signals Cooperative binding of proteins to DNA Turning genes on or off activation versus repression 3. Bacteriophage Lambda The genetic switch of lysogeny versus lytic growth Establishing lysogeny Making an efficient switch the importance of cooperativity The repressor as a gene activator DNA binding and synergy 4. Polymerase activation: NtrC and conformational changes in pre-bound polymerase 5. Promoter activation 2

3 Part II: Eukaryotic transcriptional regulation (Friday, 2 lectures; summarized, reviewed and extended upon on Friday, October 17) Although the principles utilized by eukaryotes to regulate gene transcription are the same as you will have learned from examining bacterial gene regulation, the details are both different and fascinating in their own right. Eukaryotes are also a marvelous example of how simple principles can be combined into complex regulatory schemes. We will focus on yeast as a model system to learn about how eukaryotes regulate gene activity. Before class on Friday, please read Chapter 2, pages In these two lectures, we will focus on: 1. The structure of the eukaryote RNA polymerase machinery 2. Gal4 as a model transcriptional regulator 3. The nature of the activation domain in a transcriptional regulator 4. Recruitment of the RNA polymerase by transcriptional regulators 5. The role of nucleosomal modifiers in gene regulation 6. Transcriptional repression 7. Cooperative and combinatorial control of gene activity After the next three lectures on Chromatin structure and its role in gene expression and regulation, we will review transcriptional regulation in the first hour on Friday, October 19. 3

4 RNA Processing Monday, October 13 Dale Dorsett, PhD., Doisy Research Center room 423, Ext Production of mature messenger RNA and the mrna life cycle Readings: Zorio, DAR, Bently, DL The link between RNA processing and transcription: communication works both ways. Exp Cell Res 296: Concepts: mrna capping mrna splicing reiterative splicing of long introns mrna polyadenylation The RNA Pol II C-terminal domain and coordination of RNA processing mrna transport mrna localization mrna degradation and turnover Degradation of missense mutant mrna Mechanisms of RNAi-induced degradation 4

5 Chromatin Structure and Transcription Tuesday October 14 Thursday October 16 Joel Eissenberg, Doisy Research Center, Room 421, Ext In each lecture, I ll present some background material related to the key questions and present experimental results from two research papers illustrating experimental approaches to answering these questions. Chromatin structure and gene activation I All, or nearly all of the DNA in a eukaryotic nucleus is packaged into nucleosomes. What is a nucleosome? How could the DNA-nucleosome interaction affect the ability of DNA to be used as a template for transcription? How does a transcription factor interact with a nucleosomal template? Readings: Li, G., and J. Widom (2004) Nucleosomes facilitate their own invasion Nature Structural Mol. Biol. 11: Owen-Hughes, T., and J.L. Workman (1996) Remodeling the chromatin structure of a nucleosome array by transcription factor-targeted trans-displacement of histones. EMBO J. 15: Chromatin structure and gene activation II Histones are covalently modified in vivo. These modification are correlated with various aspects of chromosome activity, including gene regulation. How is chromatin modified to facilitate gene activation? Readings: Peterson, C.L. and M.-A. Laniel (2004) Histones and histone modifications Curr. Biol 14: R Kuo, M.-H., J. Zhou, P. Jambeck, M. E. A. Churchill, and C.D. Allis (1998) Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. Genes Devel. 12:

6 Chromatin structure and repression Methylation of lysines at certain sites on certain histones is correlated with gene silencing. How are these methylated residues specifically recognized? How might histone methylation contribute to gene silencing? Readings: Jacobs, SA.A., and Sepideh Khorasanizadeh (2002) Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science 295: Nielsen, S.J., R. Schneider, U.-M. Bauer, A.J. Bannister, A. Morrison, D. O Carroll, R. Firestein, M. Cleary, T, Jenuwein, R.E. Herrera, and T. Kouzarides (2001) Rb targets histone H3 methylation and HP1 to promoters. Nature 412:

7 Friday October 17 1 st hour Review of Transcriptional Regulation Zassenhaus, Doisy Research Center, Room 663 or 603, Ext , Zassenp@slu.edu Important principles of transcriptional regulation: (1) Weak binding (2) Cooperativity (3) Combinatorial control (4) Localization of gene activators (5) Targeting by site-specific gene regulators (6) Gene activation by recruitment 7

8 Gene regulation by micrornas and RNA interference Friday October 17 2 nd hour George Eliceiri, Doisy Hall 5 th floor, Ext eliceiri@slu.edu Reading: Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: Concepts: What is RNA interference? What are micrornas? Biogenesis of micrornas Gene silencing by mrna cleavage Gene silencing by translational repression Gene silencing by heterochromatin transcription inhibition 8

9 Protein Synthesis Monday October 20 and Tuesday October 21 Yie-Hwa Chang, Ph.D., Doisy Research Center room 515, Ext Suggested readings: 1. Cell and Molecular Biology, Kleinsmith and Kish (2 nd edition), Chapter Berg, JM, Lorsch, JR (2001) Mechanism of ribosomal peptide bond formation. Science 291: Ibba, M., Soll, D. (1999) Quality control mechanism during translation. Science 286: 1893 Part I: Mechanism of protein synthesis 1. Ribosome structure 2. Protein Synthesis 2.1 mrna 2.2 trna 2.3 The initiation of protein synthesis 2.4 Peptide bond formation 2.6 Translocation 2.7 Termination of protein synthesis 2.8 Polysomes Part II: The regulation of protein synthesis Suggested readings: 1. Cell and Molecular Biology, Kleinsmith and Kish (2 nd edition), Chapter Pestova, TV, et al (2001) Molecular mechanism of translation initiation in eukaryotes. Proc. Nat. Acad. Sci. USA 98:

10 3. Gale, M., Tan, S.L., and Katze M.G. (2000) Translational control of viral gene expression in eukaryotes. Miocrobiol. Mol. Biol. Rev. 64: Translational repressors 2. Life span of mrna 3. RNAi 4. Phosphorylation 5. Availability of trna 5. Rate of termination of translation 7. Initiation factors. 10

11 Protein Folding and Quality Control; Targeted Proteolysis as a Main Regulatory Mechanism of Gene Expression Wednesday October 22 Friday October 24 (three lectures) Dorota Skowyra, PhD., Doisy Research Center room 407, Ext skowyrad@slu.edu The economics of protein synthesis, folding, and degradation: the evolutionary solutions to optimize the cost of gene expression (Thursday) Reading: (1) Yewdell, J.W. (2001): Not such a dismal science: the economics of protein synthesis, folding, degradation and antigen processing. Trends in Cell Biology, 11(7), (2) Schubet, U., Anton, LO.C., Gibbs, J., Norbury, C.C., Yewdell, J.W., Bennink, J.R. (2000): Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, Overview: Once synthesized on the ribosome, every polypeptide needs to fold into a conformation that ensures its designed function, modification and/or interaction with other proteins. Frequently, such conformation involves multiple independently folded modules and is hard to be achieved by spontaneous folding of the polypeptide itself. Indeed, in the cell, protein folding is assisted by molecular chaperones, which in multiple rounds of ATP-dependent binding and release massage the protein into its optimal shape. If this process fails, the misfolded polypeptides are recognized by cellular quality control systems and eliminated by targeted proteolysis via the ubiquitinproteasome pathway. A proteolytic pathway that recognizes and destroys abnormal proteins must be able to distinguish between completed proteins that have wrong conformations and the many growing polypeptides on ribosomes that have yet not achieved their normal folded conformation. That this is not a trivial issue is demonstrated by the observation that in normal growth conditions approximately one third of newly synthesized proteins are degraded within minutes of their synthesis (Ref 2). Is this the best evolutionary solution to the problem of optimizing the cost of a successful expression of a given gene? We will discuss this issue in class. PLEASE, READ THE SUGGESTED LITERATURE AND PREPARE FOR DISCUSSION. Problem (prepare your opinion to discuss in class): In your opinion, is the high rate of degradation of the newly synthesized proteins the best solution to the problem of optimizing the cost of a successful gene expression? Ubiquitin-dependent proteolysis as a key regulator of the biological processes, including gene expression (Friday) 11

12 Reading: (1) Selected chapters from: Glickman, M., and Ciechanover, A. (2002): The Ubiquitin- Proteasome Proteolytic Pathway: Destruction for the Sake of Construction, Physiol. Rev. 82: Chapter I: Introduction and overview, pp Chapter II: The ubiquitin conjugation machinery, pp Chapter IV: Modes of substrate recognition and regulation of the ubiquitin pathway, pp (2) Conaway, R.C., Brower, C.S., Weliky-Conaway, J. (2004) Emerging roles of ubiquitin in transcription regulation. Science 296, (read for Friday). Overview: Between the 1960s and 1980s protein degradation was a neglected area, considered to be a non-specific dead-end process. Although it was known that proteins do turn over, the large extent and specificity of this process, whereby distinct proteins have half-lives that range from a few minutes to several days, was not appreciated. The discovery of the lysosome by Christian de Duve did not significantly change this view, because it became clear that this organelle is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate specific. The discovery of the complex cascade of the ubiquitin pathway revolutionized the field. It is clear now that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of pathways during cell life and death, including the regulation of gene expression, stress and immune responses, cell cycle control and metabolic adaptation. We will discuss how the ubiquitin-mediated proteolysis contributes to the regulation of gene expression. Problem (prepare your opinion to discuss in class): how would you design an evolutionary conserved system for intracellular protein degradation which would be required to target 80% of total cellular proteins in a specific, regulated (when needed), and timely (fast) manner? 12

13 Monday October 27, 9 AM Noon Exam A total of 80 points, 5 points per lecture 13